Interleukin-13 binding proteins

ABSTRACT

The present invention encompasses IL-13 binding proteins. Specifically, the invention relates to antibodies that are chimeric, CDR grafted and humanized antibodies. Preferred antibodies have high affinity for hIL-13 and neutralize hIL-13 activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. Method of making and method of using the antibodies of the invention are also provided. The antibodies, or antibody portions, of the invention are useful for detecting hIL-13 and for inhibiting hIL-13 activity, e.g., in a human subject suffering from a disorder in which hIL-13 activity is detrimental.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 11/899,819, filedSep. 7, 2007 now U.S. Pat. No. 7,915,388, which claims the benefit ofpriority to U.S. provisional application No. 60/843,249, filed Sep. 8,2006, now expired.

FIELD OF THE INVENTION

The present invention relates to IL-13 binding proteins, andspecifically to their uses in the prevention and/or treatment of variousdiseases including asthma, allergy, COPD, fibrosis, and cancer.

REFERENCE TO JOINT RESEARCH AGREEMENT

Contents of this application are under a joint research agreemententered into by and between Protein Design Labs, Inc. and AbbottLaboratories on Dec. 14, 2005, and directed to recombinantly engineeredantibodies to IL-13.

BACKGROUND OF THE INVENTION

Human IL-13 is a 17-kDa glycoprotein cloned from activated T cells(Zurawski and de Vries 1994 Immunol Today 15 19-26), and is produced byactivated T cells of the Th2 lineage, although Th0 and Th1 CD4+ T cells,CD8+ T cells, and several non-T cell populations such as mast cells alsoproduce IL-13 (Zurawski and de Vries 1994 Immunol Today 15 19-26). Thefunction of IL-13 includes immunoglobulin isotype switching to IgE inhuman B cells (Punnonen, Aversa et al. 1993 Proc Natl Acad Sci USA 903730-4) and suppressing inflammatory cytokine production in both humanand mouse (de Waal Malefyt, Figdor et al. 1993 J Immunol 151 6370-81;Doherty, Kastelein et al. 1993 J Immunol 151 7151-60). IL-13 binds toits cell surface receptors, IL-13Ralpha1 and IL-13Ralpha2. TheIL-13Ralpha1 interacts with IL-13 with a low affinity (KD˜10 nM),followed by recruitment of IL-4Ra to form the high affinity (KD˜0.4 nM)signaling heterodimeric receptor complex (Aman, Tayebi et al. 1996 JBiol Chem 271 29265-70; Hilton, Zhang et al. 1996 Proc Natl Acad Sci USA93 497-501). The IL-4R/IL-13Ralpha1 complex is expressed on many celltypes such as B cells, monocyte/macrophages, dendritic cells,eosinophils, basophils, fibroblasts, endothelial cells, airwayepithelial cells, and airway smooth muscle cells (Graber, Gretener etal. 1998 Eur J Immunol 28 4286-98; Murata, Husain et al. 1998 IntImmunol 10 1103-10; Akaiwa, Yu et al. 2001 Cytokine 13 75-84). Ligationof the IL-13Ralpha1/IL-4R receptor complex results in activation of avariety of signal-transduction pathways including signal transducer andactivator of transcription (STAT6) and the insulin receptor substrate-2(IRS-2) pathways (Wang, Michieli et al. 1995 Blood 86 4218-27; Takeda,Kamanaka et al. 1996 J Immunol 157 3220-2). The IL-13Ralpha2 chain alonehas a high affinity (KD ˜0.25-0.4 nM) for IL-13, and functions as both adecoy receptor negatively regulating IL-13 binding (Donaldson, Whitterset al. 1998 J Immunol 161 2317-24), and a signaling receptor thatinduces TGF-b synthesis and fibrosis via AP-1 pathway in macrophages andpossibly other cell types (Fichtner-Feigl, Strober et al. 2006 Nat Med12 99-106).

Several studies conducted in preclinical animal models for asthmaindicate that IL-13 plays an important role in asthma. These datainclude resistance to asthma in the IL-13 knockout mice as well asinhibition of the asthma phenotype with IL-13 antagonists (soluble IL-13receptors, anti-IL-13 mAbs, etc.) in various mouse models (Sela 1999Harefuah 137 317-9; Wills-Karp and Chiaramonte 2003 Curr Opin Pulm Med 921-7; Wills-Karp 2004 Immunol Rev 202 175-90). Multiple studies havedemonstrated that pharmacologic administration of recombinant IL-13 tothe lungs of mice as well as guinea pigs induces airway mucushyper-secretion, eosinophilia and AHR (Grunig, Warnock et al. 1998Science 282 2261-3; Wills-Karp, Luyimbazi et al. 1998 Science 2822258-61; Kibe, Inoue et al. 2003 Am J Respir Crit Care Med 167 50-6;Vargaftig and Singer 2003 Am J Physiol Lung Cell Mol Physiol 284 L260-9;Vargaftig and Singer 2003 Am J Respir Cell Mol Biol 28 410-9). Theseeffects of IL-13 are reproduced in transgenic mouse systems with eitherconstitutive or inducible expression of IL-13 (Zhu, Homer et al. 1999 JClin Invest 103 779-88; Zhu, Lee et al. 2001 Am J Respir Crit. Care Med164 567-70; Lanone, Zheng et al. 2002 J Clin Invest 110 463-74). Chronictransgenic over-expression of IL-13 also induces subepithelial fibrosisand emphysema. Mice deficient in the IL-13 (and IL-4) signaling moleculeSTAT6 fail to develop allergen-induced AHR and mucus overproduction(Kuperman, Huang et al. 2002 Nat Med 8 885-9). Studies using solubleIL-13 receptor fusion protein (sIL-13Ralpha2Fc) have demonstrated thepivotal role of this cytokine in experimental allergen ovalbumin(OVA)-induced airway disease (Grunig, Warnock et al. 1998 Science 2822261-3; Wills-Karp, Luyimbazi et al. 1998 Science 282 2258-61; Taube,Duez et al. 2002 J Immunol 169 6482-9). Efficacy of anti-IL-13 treatmentwas also demonstrated in a chronic model of murine asthma. In additionto exhibiting features of mucus hyper-secretion and AHR, this model ofchronic asthma demonstrates several hallmarks of human disease that arelacking in the more acute models. These include eosinophilia of the lungtissue located in inter-epithelial spaces as well as smooth musclefibrosis as measured by increases in collagen deposition. The chronicasthma model is induced with repeated aerosol challenges with OVA inOVA-sensitized mice 1×/week for a total of 4 weeks. Anti-IL-13 antibodyadministered for the final 2 weeks of OVA challenges (from day 36 withefficacy readouts assessed on day 53 of study) significantly inhibitedAHR, pulmonary inflammation, goblet cell hyperplasia, mucushypersecretion, and airway fibrosis (Yang, Li et al. 2005 J PharmacolExp Ther). Moreover, therapeutic effect of IL-13 antagonist was alsodemonstrated to inhibit AHR in a primate model of asthma [Abstract,American Thoracic Society 2005].

IL-13 is implicated in the pathogenesis of human asthma as elevatedlevels of IL-13 mRNA and protein have been detected in lungs ofasthmatic patients, which correlate with severity of the disease (Huang,Xiao et al. 1995 J Immunol 155 2688-94). In addition, human IL-13genetic polymorphisms, which lead to elevated IL-13 levels, have beenidentified and are associated with asthma and atopy (Heinzmann, Mao etal. 2000 Hum Mol Genet 9 549-59; Hoerauf, Kruse et al. 2002 MicrobesInfect 4 37-42; Vercelli 2002 Curr Opin Allergy Clin Immunol 2 389-93;Heinzmann, Jerkic et al. 2003 J Allergy Clin Immunol 112 735-9; Chen,Ericksen et al. 2004 J Allergy Clin Immunol 114 553-60; Vladich,Brazille et al. 2005 J Clin Invest), and elevated IL-13 levels have beendetected in the lung of asthma patients (Huang, Xiao et al. 1995 JImmunol 155 2688-94; Arima, Umeshita-Suyama et al. 2002 J Allergy ClinImmunol 109 980-7; Berry, Parker et al. 2004 J Allergy Clin Immunol 1141106-9). A genetic linkage between IL-13 and asthma has also beendemonstrated as individuals with a polymorphism in the IL-13 gene whichcauses higher plasma IL-13 levels have an increased risk for atopy andasthma (Wills-Karp 2000 Respir Res 1 19-23).

Due to the role of human IL-13 in a variety of human disorders,therapeutic strategies have been designed to inhibit or counteract IL-13activity. In particular, antibodies that bind to, and neutralize, IL-13have been sought as a means to inhibit IL-13 activity. However, thereexists a need in the art for improved antibodies capable of bindingIL-13. Preferably the antibodies bind human IL-13. Preferably theantibodies are capable of neutralizing human IL-13. The presentinvention provides a novel family of binding proteins, CDR graftedantibodies, humanized antibodies, and fragments thereof, capable bindinghuman IL-13, binding with high affinity, and binding and neutralizinghuman IL-13.

SUMMARY OF THE INVENTION

This invention pertains to IL-13 binding proteins. Binding proteins ofthe inventions include, but are not limited to antibodies, antigenbinding portions, and other antigen binding proteins capable of bindingthe human IL-13. Further, the invention provides methods of making andusing IL-13 binding proteins.

One aspect of the invention pertains to a binding protein capable ofbinding IL-13. In a preferred embodiment, the binding protein bindshuman IL-13. Preferably the binding protein is capable of modulating abiological function of IL-13. More preferably the binding protein iscapable of neutralizing IL-13.

In one aspect of the invention, the binding protein is capable ofbinding IL-13, and preventing the binding of IL-13 to the IL-13α1receptor. In another aspect of the invention, the binding protein iscapable of binding IL-13, and preventing the binding of IL-13 to theIL-13α2 receptor. In a preferred embodiment, the binding protein iscapable of binding IL-13, and preventing the binding of IL-13 to boththe IL-13α1 receptor and the IL-13α2.

One embodiment of the invention provides an isolated antibody, orantigen binding fragment thereof, wherein said antibody, or antigenbinding fragment thereof binds human IL-13 and inhibits the binding ofsaid IL-13 to the IL-13α2 receptor in a cell surface-based receptorbinding assay with an IC₅₀ selected from the group consisting of about1.5×10⁻⁸ to 1×10⁻⁸ M, 1×10⁻⁸ to 1×10⁻⁹ M, 10⁻⁹ to 10⁻¹⁰M and 10⁻¹⁰ to10⁻¹¹M or in an ELISA-based receptor binding assay with an with an IC₅₀selected from the group consisting of about 1.8×10⁻⁸ to 1×10⁻⁸ M, 1×10⁻⁸to 1×10⁻⁹ M, 10⁻⁹ to 10⁻¹⁰M and 10⁻¹⁰ to 10⁻¹¹ M. Preferably theantibody binds human IL-13 and inhibits the binding of said IL-13 to theIL-13α2 receptor in a cell surface-based receptor binding assay with anIC₅₀ of 2.7×10⁻⁹M and in an ELISA-based receptor binding assay with anwith an IC₅₀ of 1.1×10⁻⁹ M. Preferably the antibody, or antigen bindingfragment thereof binds human IL-13 and inhibits the binding of saidIL-13 to the IL-13α2 receptor in a cell surface-based receptor bindingassay or in an ELISA-based receptor binding assay by about 70-100% at aconcentration of 100 nM. Preferably the antibody is 13C5.5. Morepreferably the antibody is not BAK502G9, mAb13.2 or MJ2-7.

In another aspect, the invention provides an isolated antibody, orantigen binding fragment thereof, wherein said antibody, or antigenbinding fragment thereof binds human IL-13 and inhibits AHR by about50%, 60%, 70%, 80%, 90% or 100% in a human IL-13 induced asthma model.Preferably, the antibody inhibits AHR by greater than 86% in a humanIL-13 induced asthma model. In another embodiment, the isolatedantibody, or antigen binding fragment thereof, binds human IL-13 andinhibits AHR by about 50%, 60%, 70%, 80% 90% or 100% and inhibits mucusproduction by about 40%, 50%, 60%, 70%, 80% 90% or 100% a human IL-13induced asthma model. Preferably the antibody is 13C5.5. More preferablythe antibody is not BAK502G9, mAb13.2 or MJ2-7.

In one embodiment, the binding protein of the invention has an on rateconstant (k_(on)) to IL-13 of at least about 10²M⁻¹s⁻¹; at least about10³M⁻¹s⁻¹; at least about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; or atleast about 10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance.Preferably, the binding protein of the invention has an on rate constant(k_(on)) to IL-13 between 10²M⁻¹s⁻¹ to 10³M⁻¹s⁻¹; between 10³M⁻¹s⁻¹ to10⁴M⁻¹s⁻¹; between 10⁴M⁻¹s⁻¹ to 10⁵M⁻¹s⁻¹; or between 10⁵M⁻¹s⁻¹ to10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance.

In another embodiment, the binding protein of the invention has an offrate constant (k_(off)) to IL-13 of at most about 10⁻³s⁻¹; at most about10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹; or at most about 10⁻⁶s⁻¹, as measured bysurface plasmon resonance. Preferably, the binding protein of theinvention has an off rate constant (k_(off)) to IL-13 of 10⁻³s⁻¹ to10⁻⁴s⁻¹; of 10⁻⁴s⁻¹ to 10⁻⁵s⁻¹; or of 10⁻⁵s⁻¹ to 10⁻⁶s⁻¹, as measured bysurface plasmon resonance.

In another embodiment, the binding protein of the invention has adissociation constant (K_(D)) to IL-13 of at most about 10⁻⁷ M; at mostabout 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at most about10⁻¹¹ M; at most about 10⁻¹² M; or at most 10⁻¹³M. Preferably, thebinding protein of the invention has a dissociation constant (K_(D)) toIL-13 of 10⁻⁷ M to 10⁻⁸ M; of 10⁻⁸ M to 10⁻⁹ M; of 10⁻⁹ M to 10⁻¹⁰ M; of10⁻¹⁰ to 10⁻¹¹ M; of 10⁻¹¹ M to 10⁻¹² M; or of 10⁻¹² to M 10⁻¹³M.

Preferably the antibody or antigen binding fragment thereof, binds IL-13with binding characteristics selected from the group consisting of: a)an on rate constant (k_(on)) between about 10⁵M⁻¹s⁻¹ to 10⁶M⁻¹s⁻¹ orabout 10⁶M⁻¹s⁻¹ to 10⁷M⁻¹s⁻¹, or b) an off rate constant (k_(off)) ofabout 10⁻⁴s⁻¹ to 10⁻⁵s⁻¹; or of about 10⁻⁵s⁻¹ to 10⁻⁶s⁻¹, as measured bysurface plasmon resonance; or c) a dissociation constant (K_(D)) ofabout 1.5×10⁻¹⁰ to 1×10⁻¹⁰ M or about 10⁻¹⁰ to 10⁻¹¹ M. Preferably theantibody, or antigen binding fragment thereof has an on rate constant(k_(on)) to IL-13 selected from the group consisting of: 6.68×10⁵M⁻¹s⁻¹,7.86×10⁵M⁻¹s⁻¹, 8.35×10⁵M⁻¹s⁻¹, 8.69×10⁵M⁻¹s⁻¹, 9.15×10⁵M⁻¹s⁻¹,1.26×10⁶M⁻¹s⁻¹, 1.7×10⁶M⁻¹s⁻¹, and 2.51×10⁶M⁻¹s⁻¹. Preferably theantibody, or antigen binding fragment thereof has an off rate constant(k_(off)) to IL-13 selected from the group consisting of: 1.23×10⁻⁴s⁻¹;1.76×10⁻⁴s⁻¹; 4.74×10⁻⁴s⁻¹; 1.91×10⁻⁵s⁻¹; 2.14×10⁻⁵s⁻¹, 3.82×10⁻⁵s⁻¹;8.81×10⁻⁵s⁻¹ and 9.65×10⁻⁵s⁻¹, as measured by surface plasmon resonance.Preferably the antibody, or antigen binding fragment thereof has adissociation constant (K_(D)) to IL-13 selected from the groupconsisting of: 1.05×10⁻¹⁰ M; 7.10×10⁻¹⁰ M; 1×10⁻¹¹ M; 2.20×10⁻¹¹ M;2.72×10⁻¹¹ M; 4.17×10⁻¹¹ M; 5.68×10⁻¹¹ M; 7.01×10⁻¹¹ M; 7.10×10⁻¹¹ M;and 9.79×10⁻¹¹ M.

One aspect of the invention pertains to binding proteins capable of aspecific epitope on IL-13. Preferably the specific epitope comprises theC-terminal Helix D region of human IL-13. More preferably, the specificepitope comprises the amino acid sequence VRDTK IEVAQ FVKDL LL HLK KLFREGR, corresponding to amino acid 104-130 of SEQ ID NO. 1. In anotheraspect the antibody or antigen binding portion, binds an epitopecomprising C-terminal Helix D region and N-terminal Helix A region ofhuman IL-13. Preferably the antibody, or antigen binding fragmentthereof binds human IL-13 such that IL-13 with said antibody, or antigenbinding fragment thereof, bound to the epitope defined by thetopographic regionsSer26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38and Lys123-Lys124-Leu125-Phe126-Arg127-Glu-128-Gly129-Arg130 of SEQ IDNo. 1 is inhibited from binding to the IL-13 receptor. Preferably theantibody, or antigen binding fragment thereof binds human IL-13 suchthat IL-13 with said antibody, or antigen binding fragment thereof,bound to the epitope defined by the topographic regionsArg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38 andLys123-Lys124-Leu125-Phe126-Arg127 of SEQ ID No. 1 is inhibited frombinding to the IL-13 receptor. Preferably the antibody, or antigenbinding fragment thereof binds human IL-13 such that IL-13 with saidantibody, or antigen binding fragment thereof, bound to the epitopedefined by the topographic regionsSer26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38and Lys123-Lys124-Leu125-Phe126-Arg127-Glu-128-Gly129-Arg130 of SEQ IDNo. 1 is inhibited from binding to the IL-13α2 receptor. More preferablythe antibody, or antigen binding fragment thereof binds human IL-13 suchthat IL-13 with said antibody, or antigen binding fragment thereof,bound to the epitope defined by the topographic regionsSer26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38and Lys123-Lys124-Leu125-Phe126-Arg127-Glu-128-Gly129-Arg130 of SEQ IDNo. 1 is inhibited from binding to the IL-13α2 receptor, provided saidantibody is not BAK502G9 or MJ2-7. Most preferably the antibody is13C5.5.

In one aspect the isolated antibody, or antigen binding fragmentthereof, binds IL-13 and prevents binding of IL-13 to the IL-13α2receptor with binding characteristics selected from the group consistingof binding to an epitope on IL-13 including Helix A and D; an on rateconstant (k_(on)) between about 10⁵M⁻¹s⁻¹ to 10⁶M⁻¹s⁻¹ or about10⁶M⁻¹s⁻¹ to 10⁷M⁻¹s⁻¹; an off rate constant (k_(off)) of about 10⁻⁴s⁻¹to 10⁻⁵s⁻¹; or of about 10⁻⁵s⁻¹ to 10⁻⁶s⁻¹, as measured by surfaceplasmon resonance; and a dissociation constant (K_(D)) of about1.5×10⁻¹⁰ to 1×10⁻¹⁰ M or about 10⁻¹⁰ to 10⁻¹¹ M. In another aspect theisolated antibody, or antigen binding fragment thereof, binds variantIL-13 and prevents binding of variant IL-13 to the IL-13α2 receptor withbinding characteristics selected from the group consisting of binding toan epitope on IL-13 including Helix A and D; an on rate constant(k_(on)) between about 10⁵M⁻¹s⁻¹ to 10⁶M⁻¹s⁻¹ or about 10⁶M⁻¹s⁻¹ to10⁷M⁻¹s⁻¹; an off rate constant (k_(off)) of about 10⁻⁴s⁻¹ to 10⁻⁵s⁻¹;or of about 10⁻⁵s¹ to 10⁻⁶s⁻¹, as measured by surface plasmon resonance;and a dissociation constant (K_(D)) of about 1.5×10⁻¹⁰ to 1×10⁻¹⁰ M orabout 10⁻¹⁰ to 10⁻¹¹ M.

In one aspect the invention binding protein capable of binding IL-13,said antigen binding domain comprising at least one CDR comprising anamino acid sequence selected from the group consisting of:

-   -   CDR-H1. X₁-X₂-X₃-X₄-X₅-X₆-X₇ (SEQ ID NO: 64), wherein;        -   X₁ is T, D, G, or S;        -   X₂ is S;        -   X₃ is D;        -   X₄ is M, S, Y, L, or H;        -   X₅ is G, W, Y, A, S, or N;        -   X₆ is V, I, or M; and        -   X₇ is D, H, S, Y, N, or G;    -   CDR-H2.        X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇ (SEQ        ID NO: 65), wherein;        -   X₁ is M, E, H, R, S, G, or L;        -   X₂ is I or not present;        -   X₃ is H, Y, A, D, S, or W;        -   X₄ is P, S, W, or G;        -   X₅ is S, G, E, or D;        -   X₆ is D, G, S, E, or N;        -   X₇ is S, Y, or G;        -   X₈ is E, N, Y, V, or R;        -   X₉ is T, I, or K;        -   X₁₀ is R, Y, I, D, or A;        -   X₁₁ is L, Y, D, or F;        -   X₁₂ is N, P, S, or D;        -   X₁₃ is Q, E, D, P, or S;        -   X₁₄ is K, M, S, T, A, or V;        -   X₁₅ is F, L, V, or M;        -   X₁₆ is K, R, or Q; and        -   X₁₇ is D, G, or S;    -   CDR-H3. X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID        NO: 66), wherein;        -   X₁ is W, T, G, Y, D, or I;        -   X₂ is R, A, S, G, or V;        -   X₃ is T, F, Y, or S;        -   X₄ is S, T, or Y;        -   X₅ is Y, F, or G;        -   X₆ is F, or Y;        -   X₇ is S, Y, I, or F;        -   X₈ is D, L, Y, or P;        -   X₉ is Y;        -   X₁₀ is G;        -   X₁₁ is Y, A, P, or E;        -   X₁₂ is F, M, S, L, or I;        -   X₁₃ is D, V, N, or K; and        -   X₁₄ is Y, or F;    -   CDR-L1.        X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇ (SEQ        ID NO: 67), wherein;        -   X₁ is K, or R;        -   X₂ is S, or A;        -   X₃ is S or T;        -   X₄ is Q, K, or I;        -   X₅ is N, S, T, G, or E;        -   X₆ is L, T, or S;        -   X₇ is L, Q, or V;        -   X₈ is Y, N, H, D, or T;        -   X₉ is S, I, or T;        -   X₁₀ is S, D, N, H, or Y;        -   X₁₁ is N, or G;        -   X₁₂ is Q;        -   X₁₃ is K, F, N, E, or S;        -   X₁₄ is N, T, or S;        -   X₁₅ is Y, or F;        -   X₁₆ is L, A, or M; and        -   X₁₇ is A, D, E, H, or N;    -   CDR-L2. X₁-X₂-X₃-X₄-X₅-X₆-X₇ (SEQ ID NO: 68), wherein;        -   X₁ is L, S, K, T, W, or Y;        -   X₂ is V, T, or A;        -   X₃ is S, or N;        -   X₄ is N, K, T, M, or R;        -   X₅ is R, K, or L;        -   X₆ is F, D, E, H, P, or A; and        -   X₇ is S, R, or P;    -   and    -   CDR-L3. X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉ (SEQ ID NO: 69), wherein;        -   X₁ is F, W, Q or A;        -   X₂ is Q or L;        -   X₃ is H, G, Y, W, or N;        -   X₄ is N, S, T, L, or Y;        -   X₅ is Y, T, S, E, or H;        -   X₆ is L, V, F, Y, N, G, P, or D;        -   X₇ is P, or, H;        -   X₈ is L, F, Y, W, or R; and        -   X₉ is T, or V.

Preferably, the antigen binding domain comprises at least one CDRcomprising an amino acid sequence selected from the group consisting of:

residues 31-35 of SEQ ID NO.: 32; residues 50-66 of SEQ ID NO.: 32;residues 99-105 of SEQ ID NO.: 32; residues 24-39 of SEQ ID NO.: 33;residues 55-61 of SEQ ID NO.: 33; residues 94-102 of SEQ ID NO.: 33;residues 31-35 of SEQ ID NO.: 34; residues 50-66 of SEQ ID NO.: 34;residues 99-105 of SEQ ID NO.: 34; residues 24-39 of SEQ ID NO.: 35;residues 55-61 of SEQ ID NO.: 35; residues 94-102 of SEQ ID NO.: 35;residues 31-35 of SEQ ID NO.: 36; residues 50-66 of SEQ ID NO.: 36;residues 99-109 of SEQ ID NO.: 36; residues 24-39 of SEQ ID NO.: 37;residues 55-61 of SEQ ID NO.: 37; residues 94-102 of SEQ ID NO.: 37;residues 31-35 of SEQ ID NO.: 38; residues 50-66 of SEQ ID NO.: 38;residues 99-109 of SEQ ID NO.: 38; residues 31-35 of SEQ ID NO.: 39;residues 50-66 of SEQ ID NO.: 39; residues 99-112 of SEQ ID NO.: 39;residues 24-39 of SEQ ID NO.: 40; residues 55-61 of SEQ ID NO.: 40;residues 94-102 of SEQ ID NO.: 40; residues 31-35 of SEQ ID NO.: 41;residues 50-66 of SEQ ID NO.: 41; residues 99-112 of SEQ ID NO.: 41;residues 31-35 of SEQ ID NO.: 42; residues 50-66 of SEQ ID NO.: 42;residues 99-100 of SEQ ID NO.: 42; residues 24-39 of SEQ ID NO.: 43;residues 55-61 of SEQ ID NO.: 43; residues 94-102 of SEQ ID NO.: 43;residues 31-35 of SEQ ID NO.: 44; residues 50-65 of SEQ ID NO.: 44;residues 98-106 of SEQ ID NO.: 44; residues 24-40 of SEQ ID NO.: 45;residues 56-62 of SEQ ID NO.: 45; residues 95-103 of SEQ ID NO.: 45;residues 31-37 of SEQ ID NO.: 46; residues 52-67 of SEQ ID NO.: 46;residues 100-112 of SEQ ID NO.: 46; residues 24-34 of SEQ ID NO.: 47;residues 50-56 of SEQ ID NO.: 47; residues 89-97 of SEQ ID NO.: 47;residues 31-37 of SEQ ID NO.: 48; residues 52-67 of SEQ ID NO.: 48;residues 100-112 of SEQ ID NO.: 48; residues 24-34 of SEQ ID NO.: 49;residues 50-56 of SEQ ID NO.: 49; residues 89-97 of SEQ ID NO.: 49;residues 31-37 of SEQ ID NO.: 50; residues 52-67 of SEQ ID NO.: 50;residues 100-112 of SEQ ID NO.: 50; residues 24-34 of SEQ ID NO.: 51;residues 50-56 of SEQ ID NO.: 51; residues 89-97 of SEQ ID NO.: 51;residues 31-35 of SEQ ID NO.: 52; residues 50-66 of SEQ ID NO.: 52;residues 99-107 of SEQ ID NO.: 52; residues 23-36 of SEQ ID NO.: 53;residues 52-58 of SEQ ID NO.: 53; residues 91-99 of SEQ ID NO.: 53;residues 31-35 of SEQ ID NO.: 54; residues 50-65 of SEQ ID NO.: 54;residues 98-107 of SEQ ID NO.: 54; residues 24-38 of SEQ ID NO.: 55;residues 54-60 of SEQ ID NO.: 55; residues 93-101 of SEQ ID NO.: 55;residues 31-35 of SEQ ID NO.: 56; residues 50-65 of SEQ ID NO.: 56;residues 98-107 of SEQ ID NO.: 56; residues 24-38 of SEQ ID NO.: 57;residues 54-60 of SEQ ID NO.: 57; residues 93-101 of SEQ ID NO.: 57;residues 31-35 of SEQ ID NO.: 58; residues 50-65 of SEQ ID NO.: 58;residues 98-107 of SEQ ID NO.: 58; residues 24-38 of SEQ ID NO.: 59;residues 54-60 of SEQ ID NO.: 59; residues 93-101 of SEQ ID NO.: 59;residues 31-35 of SEQ ID NO.: 60; residues 50-65 of SEQ ID NO.: 60;residues 98-107 of SEQ ID NO.: 60; residues 24-38 of SEQ ID NO.: 61;residues 54-60 of SEQ ID NO.: 61; residues 93-101 of SEQ ID NO.: 61;residues 31-35 of SEQ ID NO.: 62; residues 50-65 of SEQ ID NO.: 62;residues 98-107 of SEQ ID NO.: 62; residues 24-38 of SEQ ID NO.: 63;residues 54-60 of SEQ ID NO.: 63; and residues 93-101 of SEQ ID NO.: 63.

In a preferred embodiment, the binding protein comprises at least 3 CDRsare selected from a variable domain CDR set consisting of:

VH 25C8 CDR Set VH 25C8 CDR-H1 Residues 31-35 of SEQ ID NO.: 32 VH 25C8CDR-H2 Residues 50-66 of SEQ ID NO.: 32 VH 25C8 CDR-H3 Residues 99-105of SEQ ID NO.: 32 VL 25C8 CDR Set VL 25C8 CDR-L1 Residues 24-39 of SEQID NO.: 33 VL 25C8 CDR-L2 Residues 55-61 of SEQ ID NO.: 33 VL 25C8CDR-L3 Residues 94-102 of SEQ ID NO.: 33 VH 9C11 CDR Set VH 9C11 CDR-H1Residues 31-35 of SEQ ID NO.: 34 VH 9C11 CDR-H2 Residues 50-66 of SEQ IDNO.: 34 VH 9C11 CDR-H3 Residues 99-105 of SEQ ID NO.: 34 VL 9C11 CDR SetVL 9C11 CDR-L1 Residues 24-39 of SEQ ID NO.: 35 VL 9C11 CDR-L2 Residues55-61 of SEQ ID NO.: 35 VL 9C11 CDR-L3 Residues 94-102 of SEQ ID NO.: 35VH 21D9 CDR Set VH 21D9 CDR-H1 Residues 31-35 of SEQ ID NO.: 36 VH 21D9CDR-H2 Residues 50-66 of SEQ ID NO.: 36 VH 21D9 CDR-H3 Residues 99-109of SEQ ID NO.: 36 VL 21D9 CDR Set VL 21D9 CDR-L1 Residues 24-39 of SEQID NO.: 37 VL 21D9 CDR-L2 Residues 55-61 of SEQ ID NO.: 37 VL 21D9CDR-L3 Residues 94-102 of SEQ ID NO.: 37 VH 22D10 CDR Set VH 22D10CDR-H1 Residues 31-35 of SEQ ID NO.: 38 VH 22D10 CDR-H2 Residues 50-66of SEQ ID NO.: 38 VH 22D10 CDR-H3 Residues 99-109 of SEQ ID NO.: 38 VL22D10 CDR Set VL 22D10 CDR-L1 Residues 24-39 of SEQ ID NO.: 37 VL 22D10CDR-L2 Residues 55-61 of SEQ ID NO.: 37 VL 22D10 CDR-L3 Residues 94-102of SEQ ID NO.: 37 VH 5F1 CDR Set VH 5F1 CDR-H1 Residues 31-35 of SEQ IDNO.: 39 VH 5F1 CDR-H2 Residues 50-66 of SEQ ID NO.: 39 VH 5F1 CDR-H3Residues 99-112 of SEQ ID NO.: 39 VL 5F1 CDR Set VL 5F1 CDR-L1 Residues24-39 of SEQ ID NO.: 40 VL 5F1 CDR-L2 Residues 55-61 of SEQ ID NO.: 40VL 5F1 CDR-L3 Residues 94-102 of SEQ ID NO.: 40 VH 5G1 CDR Set VH 5G1CDR-H1 Residues 31-35 of SEQ ID NO.: 41 VH 5G1 CDR-H2 Residues 50-66 ofSEQ ID NO.: 41 VH 5G1 CDR-H3 Residues 99-112 of SEQ ID NO.: 41 VL 5G1CDR Set VL 5G1 CDR-L1 Residues 24-39 of SEQ ID NO.: 40 VL 5G1 CDR-L2Residues 55-61 of SEQ ID NO.: 40 VL 5G1 CDR-L3 Residues 94-102 of SEQ IDNO.: 40 VH 3H7 CDR Set VH 3H7 CDR-H1 Residues 31-35 of SEQ ID NO.: 42 VH3H7 CDR-H2 Residues 50-66 of SEQ ID NO.: 42 VH 3H7 CDR-H3 Residues99-100 of SEQ ID NO.: 42 VL 3H7 CDR Set VL 3H7 CDR-L1 Residues 24-39 ofSEQ ID NO.: 43 VL 3H7 CDR-L2 Residues 55-61 of SEQ ID NO.: 43 VL 3H7CDR-L3 Residues 94-102 of SEQ ID NO.: 43 VH 14B2 CDR Set VH 14B2 CDR-H1Residues 31-35 of SEQ ID NO.: 44 VH 14B2 CDR-H2 Residues 50-65 of SEQ IDNO.: 44 VH 14B2 CDR-H3 Residues 98-106 of SEQ ID NO.: 44 VL 14B2 CDR SetVL 14B2 CDR-L1 Residues 24-40 of SEQ ID NO.: 45 VL 14B2 CDR-L2 Residues56-62 of SEQ ID NO.: 45 VL 14B2 CDR-L3 Residues 95-103 of SEQ ID NO.: 45VH 13C5 CDR Set VH 13C5 CDR-H1 Residues 31-37 of SEQ ID NO.: 46 VH 13C5CDR-H2 Residues 52-67 of SEQ ID NO.: 46 VH 13C5 CDR-H3 Residues 100-112of SEQ ID NO.: 46 VL 13C5 CDR Set VL 13C5 CDR-L1 Residues 24-34 of SEQID NO.: 47 VL 13C5 CDR-L2 Residues 50-56 of SEQ ID NO.: 47 VL 13C5CDR-L3 Residues 89-97 of SEQ ID NO.: 47 VH 29G5 CDR Set VH 29G5 CDR-H1Residues 31-37 of SEQ ID NO.: 48 VH 29G5 CDR-H2 Residues 52-67 of SEQ IDNO.: 48 VH 29G5 CDR-H3 Residues 100-112 of SEQ ID NO.: 48 VL 29G5 CDRSet VL 29G5 CDR-L1 Residues 24-34 of SEQ ID NO.: 49 VL 29G5 CDR-L2Residues 50-56 of SEQ ID NO.: 49 VL 29G5 CDR-L3 Residues 89-97 of SEQ IDNO.: 49 VH 33C3 CDR Set VH 33C3 CDR-H1 Residues 31-37 of SEQ ID NO.: 50VH 33C3 CDR-H2 Residues 52-67 of SEQ ID NO.: 50 VH 33C3 CDR-H3 Residues100-112 of SEQ ID NO.: 50 VL 33C3 CDR Set VL 33C3 CDR-L1 Residues 24-34of SEQ ID NO.: 51 VL 33C3 CDR-L2 Residues 50-56 of SEQ ID NO.: 51 VL33C3 CDR-L3 Residues 89-97 of SEQ ID NO.: 51 VH 4A8 CDR Set VH 4A8CDR-H1 Residues 31-35 of SEQ ID NO.: 52 VH 4A8 CDR-H2 Residues 50-66 ofSEQ ID NO.: 52 VH 4A8 CDR-H3 Residues 99-107 of SEQ ID NO.: 52 VL 4A8CDR Set VL 4A8 CDR-L1 Residues 23-36 of SEQ ID NO.: 53 VL 4A8 CDR-L2Residues 52-58 of SEQ ID NO.: 53 VL 4A8 CDR-L3 Residues 91-99 of SEQ IDNO.: 53 VH 1B6 CDR Set VH 1B6 CDR-H1 Residues 31-35 of SEQ ID NO.: 54 VH1B6 CDR-H2 Residues 50-65 of SEQ ID NO.: 54 VH 1B6 CDR-H3 Residues98-107 of SEQ ID NO.: 54 VL 1B6 CDR Set VL 1B6 CDR-L1 Residues 24-38 ofSEQ ID NO.: 55 VL 1B6 CDR-L2 Residues 54-60 of SEQ ID NO.: 55 VL 1B6CDR-L3 Residues 93-101 of SEQ ID NO.: 55 VH 3E5 CDR Set VH 3E5 CDR-H1Residues 31-35 of SEQ ID NO.: 56 VH 3E5 CDR-H2 Residues 50-65 of SEQ IDNO.: 56 VH 3E5 CDR-H3 Residues 98-107 of SEQ ID NO.: 56 VL 3E5 CDR SetVL 3E5 CDR-L1 Residues 24-38 of SEQ ID NO.: 57 VL 3E5 CDR-L2 Residues54-60 of SEQ ID NO.: 57 VL 3E5 CDR-L3 Residues 93-101 of SEQ ID NO.: 57VH 6C8 CDR Set VH 6C8 CDR-H1 Residues 31-35 of SEQ ID NO.: 58 VH 6C8CDR-H2 Residues 50-65 of SEQ ID NO.: 58 VH 6C8 CDR-H3 Residues 98-107 ofSEQ ID NO.: 58 VL 6C8 CDR Set VL 6C8 CDR-L1 Residues 24-38 of SEQ IDNO.: 59 VL 6C8 CDR-L2 Residues 54-60 of SEQ ID NO.: 59 VL 6C8 CDR-L3Residues 93-101 of SEQ ID NO.: 59 VH 5D3 CDR Set VH 5D3 CDR-H1 Residues31-35 of SEQ ID NO.: 60 VH 5D3 CDR-H2 Residues 50-65 of SEQ ID NO.: 60VH 5D3 CDR-H3 Residues 98-107 of SEQ ID NO.: 60 VL 5D3 CDR Set VL 5D3CDR-L1 Residues 24-38 of SEQ ID NO.: 61 VL 5D3 CDR-L2 Residues 54-60 ofSEQ ID NO.: 61 VL 5D3 CDR-L3 Residues 93-101 of SEQ ID NO.: 61 VH 8B6CDR Set VH 8B6 CDR-H1 Residues 31-35 of SEQ ID NO.: 62 VH 8B6 CDR-H2Residues 50-65 of SEQ ID NO.: 62 VH 8B6 CDR-H3 Residues 98-107 of SEQ IDNO.: 62 VL 8B6 CDR Set VL 8B6 CDR-L1 Residues 24-38 of SEQ ID NO.: 63 VL8B6 CDR-L2 Residues 54-60 of SEQ ID NO.: 63 VL 8B6 CDR-L3 Residues93-101 of SEQ ID NO.: 63Preferably the binding protein comprising at least two variable domainCDR sets. Preferably at least two variable domain CDR sets are selectedfrom a group consisting of:

VH 25C8 CDR Set & VL 25C8 CDR Set;

VH 9C11 CDR Set & VL 9C11 CDR Set;

VH 21D9 CDR Set & VL 21D9 CDR Set;

VH 22D10 CDR Set & VL 22D10 CDR Set;

VH 5F1 CDR Set & VL 5F1 CDR Set;

VH 5G1 CDR Set & VL 5G1 CDR Set;

VH 3H7 CDR Set & VL 3H7 CDR Set;

VH 14B2 CDR Set & VL 14B2 CDR Set;

VH 13C5 CDR Set & VL 13C5 CDR Set;

VH 29G5 CDR Set & VL 29G5 CDR Set;

VH 33C3 CDR Set & VL 33C3 CDR Set;

VH 4A8 CDR Set & VL 4A8 CDR Set;

VH 1B6 CDR Set & VL 1B6 CDR Set;

VH 3E5CDR Set & VL 3E5 CDR Set;

VH 6C8 CDR Set & VL 6C8 CDR Set;

VH 5D3 CDR Set & VL 5D3 CDR Set; and

VH 8B6 CDR Set & VL 8B6 CDR Set.

In another embodiment the binding protein disclosed above furthercomprises a human acceptor framework. Preferably the human acceptorframework comprises an amino acid sequence selected from the groupconsisting of:

SEQ ID NO.: 6 SEQ ID NO.: 7 SEQ ID NO.: 8 SEQ ID NO.: 9 SEQ ID NO.: 10SEQ ID NO.: 11 SEQ ID NO.: 12 SEQ ID NO.: 13 SEQ ID NO.: 14 SEQ ID NO.:15 SEQ ID NO.: 16 SEQ ID NO.: 17 SEQ ID NO.: 18 SEQ ID NO.: 19 SEQ IDNO.: 20 SEQ ID NO.: 21 SEQ ID NO.: 22 SEQ ID NO.: 23 SEQ ID NO.: 24 SEQID NO.: 25 SEQ ID NO.: 26 SEQ ID NO.: 27 SEQ ID NO.: 28 SEQ ID NO.: 29SEQ ID NO.: 30 AND SEQ ID NO.: 31

In a preferred embodiment the binding protein is a CDR grafted antibodyor antigen binding portion thereof capable of binding IL-13. Preferablythe CDR grafted antibody or antigen binding portion thereof comprise oneor more CDRs disclosed above. Preferably the CDR grafted antibody orantigen binding portion thereof comprises a human acceptor framework.More preferably the human acceptor framework is any one of the humanacceptor frameworks disclosed above.

In a preferred embodiment the binding protein is a humanized antibody orantigen binding portion thereof capable of binding IL-13. Preferably thehumanized antibody or antigen binding portion thereof comprise one ormore CDRs disclosed above incorporated into a human antibody variabledomain of a human acceptor framework. Preferably the human antibodyvariable domain is a consensus human variable domain. More preferablythe human acceptor framework comprises at least one Framework Regionamino acid substitution at a key residue, wherein the key residue isselected from the group consisting of a residue adjacent to a CDR; aglycosylation site residue; a rare residue; a residue capable ofinteracting with human IL-13; a residue capable of interacting with aCDR; a canonical residue; a contact residue between heavy chain variableregion and light chain variable region; a residue within a Vernier zone;and a residue in a region that overlaps between a Chothia-definedvariable heavy chain CDR1 and a Kabat-defined first heavy chainframework. Preferably the human acceptor framework human acceptorframework comprises at least one Framework Region amino acidsubstitution, wherein the amino acid sequence of the framework is atleast 65% identical to the sequence of said human acceptor framework andcomprises at least 70 amino acid residues identical to said humanacceptor framework. Preferably the Framework Region amino acidsubstitution at a key residue is selected from the group consisting of2L, 15L, 22L, 41L, 42L, 44L, 49L, 50L, 51L, 62L, 71L, 73L, 10H, 44H,46H, 48H, 67H, 68H, 70H, 72H, 74H, 76H, 83H, 84H, 86H, 87H, and 97H.

In a preferred embodiment the binding protein is a humanized antibody orantigen binding portion thereof capable of binding IL-13. Preferably thehumanized antibody, or antigen binding portion, thereof comprises one ormore CDRs disclosed above. More preferably the humanized antibody, orantigen binding portion, thereof comprises three or more CDRs disclosedabove. Most preferably the humanized antibody, or antigen bindingportion, thereof comprises six CDRs disclosed above.

In another embodiment of the claimed invention, the humanized antibodyor antigen binding portion thereof comprises at least one variabledomain having an amino acid sequence selected from the group selectedfrom the group consisting of;

SEQ ID NO.: 70 SEQ ID NO.: 71 SEQ ID NO.: 72 SEQ ID NO.: 73 SEQ ID NO.:74 SEQ ID NO.: 75 SEQ ID NO.: 76 SEQ ID NO.: 77 SEQ ID NO.: 78 SEQ IDNO.: 79 SEQ ID NO.: 80 SEQ ID NO.: 81 SEQ ID NO.: 82 SEQ ID NO.: 83 SEQID NO.: 84 SEQ ID NO.: 85 SEQ ID NO.: 92 SEQ ID NO.: 93 and SEQ ID NO.:94.

More preferably the humanized antibody or antigen binding portionthereof comprises two variable domains selected from the group disclosedabove. More preferably binding protein comprises two variable domains,wherein said two variable domains have amino acid sequences selectedfrom the group consisting of;

SEQ ID NO.:70 & SEQ ID NO.:71,

SEQ ID NO.:72 & SEQ ID NO.:73,

SEQ ID NO.:74 & SEQ ID NO.:75,

SEQ ID NO.:76 & SEQ ID NO.:77,

SEQ ID NO.:78 & SEQ ID NO.:79,

SEQ ID NO.:80 & SEQ ID NO.:81,

SEQ ID NO.:82 & SEQ ID NO.:83,

SEQ ID NO.:84 & SEQ ID NO.:85

SEQ ID NO.:80 & SEQ ID NO.:92,

SEQ ID NO.:80 & SEQ ID NO.:93, AND

SEQ ID NO.:80 & SEQ ID NO.:94.

One embodiment of the invention provides an antibody constructcomprising any one of the binding proteins disclosed above and a linkerpolypeptide or an immunoglobulin. In a preferred embodiment the antibodyconstruct is selected from the group consisting of an immunoglobulinmolecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, adisulfide linked Fv, a scFv, a single domain antibody, a diabody, amultispecific antibody, a dual specific antibody, and a bispecificantibody. In a preferred embodiment the antibody construct comprises aheavy chain immunoglobulin constant domain selected from the groupconsisting of a human IgM constant domain, a human IgG1 constant domain,a human IgG2 constant domain, a human IgG3 constant domain, a human IgG4constant domain, a human IgE constant domain, and a human IgA constantdomain. More preferably, the antibody construct comprises SEQ ID NO.:2;SEQ ID NO.:3; SEQ ID NO.:4; and SEQ ID NO.:5. In another embodiment theinvention provides an antibody conjugate comprising an the antibodyconstruct disclosed above and an agent an agent selected from the groupconsisting of; an immunoadhension molecule, an imaging agent, atherapeutic agent, and a cytotoxic agent. In a preferred embodiment theimaging agent selected from the group consisting of a radiolabel, anenzyme, a fluorescent label, a luminescent label, a bioluminescentlabel, a magnetic label, and biotin. More preferably the imaging agentis a radiolabel selected from the group consisting of: ³H, ¹⁴C, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. In a preferredembodiment the therapeutic or cytotoxic agent is selected from the groupconsisting of; an anti-metabolite, an alkylating agent, an antibiotic, agrowth factor, a cytokine, an anti-angiogenic agent, an anti-mitoticagent, an anthracycline, toxin, and an apoptotic agent.

In another embodiment the antibody construct is glycosylated. Preferablythe glycosylation is a human glycosylation pattern.

In another embodiment binding protein, antibody construct or antibodyconjugate disclosed above exists as a crystal. Preferably the crystal isa carrier-free pharmaceutical controlled release crystal. In a preferredembodiment the crystallized binding protein, crystallized antibodyconstruct or crystallized antibody conjugate has a greater half life invivo than its soluble counterpart. In another preferred embodiment thecrystallized binding protein, crystallized antibody construct orcrystallized antibody conjugate retains biological activity aftercrystallization.

One aspect of the invention pertains to a DVD binding protein comprisingbinding proteins capable of binding IL-13. Preferably the DVD bindingprotein is capable of binding IL-13 and a second target. The secondtarget is selected from the group consisting of CSF1 (MCSF), CSF2(GM-CSF), CSF3 (GCSF), FGF2, IFNα1, IFNγ, histamine and histaminereceptors, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12α, IL-12β, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,KITLG, PDGFB, IL-2Rα, IL-4R, IL-5Rα, IL-8Rα, IL-8Rβ, IL-12Rβ1, IL-12Rβ2,IL-13Rα1, IL-13Rα2, IL-18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7,CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1,CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1,GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNFSF6, YY1,CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase. Morepreferably, the DVD protein is capable of recognizing IL-13 and IL-1β,IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHRagonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; or IL-13and ADAM8. Most preferably, the DVD protein is capable of binding IL-13and TNFα.

One aspect of the invention pertains to an isolated nucleic acidencoding any one of the binding protein, antibody construct or antibodyconjugate disclosed above. A further embodiment provides a vectorcomprising the isolated nucleic acid disclosed above wherein said vectoris selected from the group consisting of pcDNA; pTT (Durocher et al.,Nucleic Acids Research 2002, Vol 30, No. 2); pTT3 (pTT with additionalmultiple cloning site; pEFBOS (Mizushima, S, and Nagata, S., (1990)Nucleic acids Research Vol 18, No. 17); pBV; pJV; and pBJ.

In another aspect a host cell is transformed with the vector disclosedabove. Preferably the host cell is a prokaryotic cell. More preferablythe host cell is E. Coli. In a related embodiment the host cell is aneukaryotic cell. Preferably the eukaryotic cell is selected from thegroup consisting of protist cell, animal cell, plant cell and fungalcell. More preferably the host cell is a mammalian cell including, butnot limited to, CHO and COS; or a fungal cell such as Saccharomycescerevisiae; or an insect cell such as Sf9.

Another aspect of the invention provides a method of producing a bindingprotein that binds IL-13, comprising culturing any one of the host cellsdisclosed above in a culture medium under conditions sufficient toproduce a binding protein that binds IL-13. Another embodiment providesa binding protein produced according to the method disclosed above.

One embodiment provides a composition for the release of a bindingprotein wherein the composition comprises a formulation which in turncomprises a crystallized binding protein, crystallized antibodyconstruct or crystallized antibody conjugate as disclosed above and aningredient; and at least one polymeric carrier. Preferably the polymericcarrier is a polymer selected from one or more of the group consistingof: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid),poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly(caprolactone), poly (dioxanone); poly (ethylene glycol), poly((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolyeaccharides, blends and copolymers thereof. Preferably theingredient is selected from the group consisting of albumin, sucrose,trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin,methoxypolyethylene glycol and polyethylene glycol. Another embodimentprovides a method for treating a mammal comprising the step ofadministering to the mammal an effective amount of the compositiondisclosed above.

The invention also provides a pharmaceutical composition comprising abinding protein, antibody construct or antibody conjugate as disclosedabove and a pharmaceutically acceptable carrier. In a further embodimentthe pharmaceutical composition comprises at least one additionaltherapeutic agent for treating a disorder in which IL-13 activity isdetrimental. Preferably the additional agent is selected from the groupconsisting of: Therapeutic agent, imaging agent, cytotoxic agent,angiogenesis inhibitors (including but not limited to anti-VEGFantibodies or VEGF-trap); kinase inhibitors (including but not limitedto KDR and TIE-2 inhibitors); co-stimulation molecule blockers(including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig,anti-CD20); adhesion molecule blockers (including but not limited toanti-LFA-1 Abs, anti-E/L selectin Abs, small molecule inhibitors);anti-cytokine antibody or functional fragment thereof (including but notlimited to anti-IL-18, anti-TNF, anti-IL-6/cytokine receptorantibodies); methotrexate; cyclosporin; rapamycin; FK506; detectablelabel or reporter; a TNF antagonist; an antirheumatic; a musclerelaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), ananalgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteriod, an anabolic steroid, an erythropoietin, an immunization,an immunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog, a cytokine, and a cytokineantagonist.

In another aspect, the invention provides a method for inhibiting humanIL-13 activity comprising contacting human IL-13 with a binding proteindisclosed above such that human IL-13 activity is inhibited. In arelated aspect the invention provides a method for inhibiting humanIL-13 activity in a human subject suffering from a disorder in whichIL-13 activity is detrimental, comprising administering to the humansubject a binding protein disclosed above such that human IL-13 activityin the human subject is inhibited and treatment is achieved.

In another aspect, the invention provides a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing anIL-13-associated disorder, in a subject. The method includes:administering to the subject an IL-13 binding agent (particularly anantagonist), e.g., an anti-IL-13 antibody or fragment thereof asdescribed herein, in an amount sufficient to treat or prevent theIL-13-associated disorder. The IL-13 antagonist, e.g., the anti-IL-13antibody or fragment thereof, can be administered to the subject, aloneor in combination with other therapeutic modalities as described herein.

In one embodiment, the subject is a mammal, e.g., a human suffering fromone or more IL-13-associated disorders, including, e.g., respiratorydisorders (e.g., asthma (e.g., allergic and nonallergic asthma), chronicobstructive pulmonary disease (COPD), and other conditions involvingairway inflammation, eosinophilia, fibrosis and excess mucus production;atopic disorders (e.g., atopic dermatitis and allergic rhinitis);inflammatory and/or autoimmune conditions of, the skin, gastrointestinalorgans (e.g., inflammatory bowel diseases (IBD), such as ulcerativecolitis and/or Crohn's disease), and liver (e.g., cirrhosis, fibrosis);scleroderma; tumors or cancers, e.g., Hodgkin's lymphoma as describedherein. Accordingly, the disclosure includes the use of an IL-13 bindingagent (such as an anti-IL-13 antibody or fragment thereof describedherein) for a treatment described herein and the use of an IL-13 bindingagent (such as an anti-IL-13 antibody or fragment thereof describedherein) for preparing a medicament for a treatment described herein.Examples of IL-13-associated disorders include, but are not limited to,a disorder chosen from one or more of: respiratory disorders, e.g.,asthma (e.g., allergic and nonallergic asthma (e.g., asthma due toinfection with, e.g., respiratory syncytial virus (RSV), e.g., inyounger children)), chronic obstructive pulmonary disease (COPD), andother conditions involving airway inflammation, eosinophilia, fibrosisand excess mucus production, e.g., cystic fibrosis and pulmonaryfibrosis; atopic disorders, e.g., resulting from an increasedsensitivity to IL-13 (e.g., atopic dermatitis, urticaria, eczema,allergic rhinitis, and allergic enterogastritis); inflammatoyv and/orautoimmune conditions of, the skin (e.g., atopic dermatitis),gastrointestinal organs (e.g., inflammatory bowel diseases (IBD), suchas ulcerative colitis and/or Crohn's disease), liver (e.g., cirrhosis,hepatocellular carcinoma), and scleroderma; tumors or cancers (e.g.,soft tissue or solid tumors), such as leukemia, glioblastoma, andlymphoma, e.g., Hodgkin's lymphoma; viral infections (e.g., fromHTLV-1); fibrosis of other organs, e.g., fibrosis of the liver, (e.g.,fibrosis caused by a hepatitis B and/or C virus); and suppression ofexpression of protective type 1 immune responses, (e.g., duringvaccination), as described herein.

In other embodiments, this application provides a method of treating(e.g., reducing, ameliorating) or preventing one or more symptomsassociated with a respiratory disorder, e.g., asthma (e.g., allergic andnonallergic asthma); allergies; chronic obstructive pulmonary disease(COPD); a condition involving airway inflammation, eosinophilia,fibrosis and excess mucus production, e.g., cystic fibrosis andpulmonary fibrosis. For example, symptoms of asthma include, but are notlimited to, wheezing, shortness of breath, bronchoconstriction, airwayhyperreactivity, decreased lung capacity, fibrosis, airway inflammation,and mucus production. The method comprises administering to the subjectan IL-13 antagonist, e.g., an IL-13 antibody or a fragment thereof, inan amount sufficient to treat (e.g., reduce, ameliorate) or prevent oneor more symptoms. The IL-13 antibody can be administered therapeuticallyor prophylactically, or both. The IL-13 antagonist, e.g., the anti-IL-13antibody, or fragment thereof, can be administered to the subject, aloneor in combination with other therapeutic modalities as described herein.Preferably, the subject is a mammal, e.g., a human suffering from anIL-13-associated disorder as described herein.

In another aspect, this application provides a method for detecting thepresence of IL-13 in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, biopsy). The subject method can be used todiagnose a disorder, e.g., an immune cell-associated disorder. Themethod includes: (i) contacting the sample or a control sample with theanti-IL-13 antibody or fragment thereof as described herein; and (ii)detecting formation of a complex between the anti-IL-13 antibody orfragment thereof, and the sample or the control sample, wherein astatistically significant change in the formation of the complex in thesample relative to the control sample is indicative of the presence ofthe IL-13 in the sample.

In yet another aspect, this application provides a method for detectingthe presence of IL-13 in vivo (e.g., in viva imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., anIL-13-associated disorder. The method includes: (i) administering theanti-IL-13 antibody or fragment thereof as described herein to a subjector a control subject under conditions that allow binding of the antibodyor fragment to IL-13; and (ii) detecting formation of a complex betweenthe antibody or fragment and IL-13, wherein a statistically significantchange in the formation of the complex in the subject relative to thecontrol subject is indicative of the presence of IL-13.

In another aspect, the binding proteins of the invention are useful fortreating a disorder selected from the group consisting of arthritis,osteoarthritis, juvenile chronic arthritis, septic arthritis, Lymearthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,systemic lupus erythematosus, Crohn's disease, ulcerative colitis,inflammatory bowel disease, insulin dependent diabetes mellitus,thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, graft versus host disease, organ transplant rejection,acute or chronic immune disease associated with organ transplantation,sarcoidosis, atherosclerosis, disseminated intravascular coagulation,Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatiguesyndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,microscopic vasculitis of the kidneys, chronic active hepatitis,uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia,infectious diseases, parasitic diseases, acquired immunodeficiencysyndrome, acute transverse myelitis, Huntington's chorea, Parkinson'sdisease, Alzheimer's disease, stroke, primary biliary cirrhosis,hemolytic anemia, malignancies, heart failure, myocardial infarction,Addison's disease, sporadic, polyglandular deficiency type I andpolyglandular deficiency type II, Schmidt's syndrome, adult (acute)respiratory distress syndrome, alopecia, alopecia areata, seronegativearthopathy, arthropathy, Reiter's disease, psoriatic arthropathy,ulcerative colitic arthropathy, enteropathic synovitis, chlamydia,yersinia and salmonella associated arthropathy, spondyloarthopathy,atheromatous disease/arteriosclerosis, atopic allergy, autoimmunebullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,linear IgA disease, autoimmune haemolytic anaemia, Coombs positivehaemolytic anaemia, acquired pernicious anaemia, juvenile perniciousanaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneouscandidiasis, giant cell arteritis, primary sclerosing hepatitis,cryptogenic autoimmune hepatitis, Acquired Immunodeficiency DiseaseSyndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B,Hepatitis C, common varied immunodeficiency (common variablehypogammaglobulinaemia), dilated cardiomyopathy, female infertility,ovarian failure, premature ovarian failure, fibrotic lung disease,cryptogenic fibrosing alveolitis, post-inflammatory interstitial lungdisease, interstitial pneumonitis, connective tissue disease associatedinterstitial lung disease, mixed connective tissue disease associatedlung disease, systemic sclerosis associated interstitial lung disease,rheumatoid arthritis associated interstitial lung disease, systemiclupus erythematosus associated lung disease,dermatomyositis/polymyositis associated lung disease, Sjögren's diseaseassociated lung disease, ankylosing spondylitis associated lung disease,vasculitic diffuse lung disease, haemosiderosis associated lung disease,drug-induced interstitial lung disease, fibrosis, radiation fibrosis,bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocyticinfiltrative lung disease, postinfectious interstitial lung disease,gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis(classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type Binsulin resistance with acanthosis nigricans, hypoparathyroidism, acuteimmune disease associated with organ transplantation, chronic immunedisease associated with organ transplantation, osteoarthrosis, primarysclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathicleucopaenia, autoimmune neutropaenia, renal disease NOS,glomerulonephritides, microscopic vasulitis of the kidneys, lymedisease, discoid lupus erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympatheticophthalmia, pulmonary hypertension secondary to connective tissuedisease, Goodpasture's syndrome, pulmonary manifestation ofpolyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis,Still's disease, systemic sclerosis, Sjörgren's syndrome, Takayasu'sdisease/arteritis, autoimmune thrombocytopaenia, idiopathicthrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrousautoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmunehypothyroidism, primary myxoedema, phacogenic uveitis, primaryvasculitis, vitiligo acute liver disease, chronic liver diseases,alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis,idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholicSteatohepatitis, allergy and asthma, group B streptococci (GBS)infection, mental disorders (e.g., depression and schizophrenia), Th2Type and Th1 Type mediated diseases, acute and chronic pain (differentforms of pain), and cancers such as lung, breast, stomach, bladder,colon, pancreas, ovarian, prostate and rectal cancer and hematopoieticmalignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis,acute and chronic parasitic or infectious processes, acute leukemia,acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acuteor chronic bacterial infection, acute pancreatitis, acute renal failure,adenocarcinomas, aerial ectopic beats, AIDS dementia complex,alcohol-induced hepatitis, allergic conjunctivitis, allergic contactdermatitis, allergic rhinitis, allograft rejection, alpha-1-antitrypsindeficiency, amyotrophic lateral sclerosis, anemia, angina pectoris,anterior horn cell degeneration, anti cd3 therapy, antiphospholipidsyndrome, anti-receptor hypersensitivity reactions, aordic andperipheral aneuryisms, aortic dissection, arterial hypertension,arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation(sustained or paroxysmal), atrial flutter, atrioventricular block, Bcell lymphoma, bone graft rejection, bone marrow transplant (BMT)rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiacarrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy,cardiopulmonary bypass inflammation response, cartilage transplantrejection, cerebellar cortical degenerations, cerebellar disorders,chaotic or multifocal atrial tachycardia, chemotherapy associateddisorders, chromic myelocytic leukemia (CML), chronic alcoholism,chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL),chronic obstructive pulmonary disease (COPD), chronic salicylateintoxication, colorectal carcinoma, congestive heart failure,conjunctivitis, contact dermatitis, cor pulmonale, coronary arterydisease, Creutzfeldt-Jakob disease, culture negative sepsis, cysticfibrosis, cytokine therapy associated disorders, Dementia pugilistica,demyelinating diseases, dengue hemorrhagic fever, dermatitis,dermatologic conditions, diabetes, diabetes mellitus, diabeticateriosclerotic disease, Diffuse Lewy body disease, dilated congestivecardiomyopathy, disorders of the basal ganglia, Down's Syndrome inmiddle age, drug-induced movement disorders induced by drugs which blockCNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis,endocarditis, endocrinopathy, epiglottitis, epstein-barr virusinfection, erythromelalgia, extrapyramidal and cerebellar disorders,familial hematophagocytic lymphohistiocytosis, fetal thymus implantrejection, Friedreich's ataxia, functional peripheral arterialdisorders, fungal sepsis, gas gangrene, gastric ulcer, glomerularnephritis, graft rejection of any organ or tissue, gram negative sepsis,gram positive sepsis, granulomas due to intracellular organisms, hairycell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hayfever, heart transplant rejection, hemachromatosis, hemodialysis,hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIVneuropathy, Hodgkin's disease, hyperkinetic movement disorders,hypersensitity reactions, hypersensitivity pneumonitis, hypertension,hypokinetic movement disorders, hypothalamic-pituitary-adrenal axisevaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis,antibody mediated cytotoxicity, Asthenia, infantile spinal muscularatrophy, inflammation of the aorta, influenza a, ionizing radiationexposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusioninjury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinalmuscular atrophy, Kaposi's sarcoma, kidney transplant rejection,legionella, leishmaniasis, leprosy, lesions of the corticospinal system,lipedema, liver transplant rejection, lymphederma, malaria, malignamtLymphoma, malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmultisystem disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of theabdominal aorta and its branches, occulsive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue, Acute coronary syndromes,Acute Idiopathic Polyneuritis, Acute Inflammatory DemyelinatingPolyradiculoneuropathy, Acute ischemia, Adult Still's Disease, Alopeciaareata, Anaphylaxis, Anti-Phospholipid Antibody Syndrome, Aplasticanemia, Arteriosclerosis, Atopic eczema, Atopic dermatitis, Autoimmunedermatitis, Autoimmune disorder associated with Streptococcus infection,Autoimmune Enteropathy, Autoimmune hearingloss, AutoimmuneLymphoproliferative Syndrome (ALPS), Autoimmune myocarditis, Autoimmunepremature ovarian failure, Blepharitis, Bronchiectasis, Bullouspemphigoid, Cardiovascular Disease, Catastrophic AntiphospholipidSyndrome, Celiac Disease, Cervical Spondylosis, Chronic ischemia,Cicatricial pemphigoid, Clinically isolated Syndrome (CIS) with Risk forMultiple Sclerosis, Conjunctivitis, Childhood Onset PsychiatricDisorder, Chronic obstructive pulmonary disease (COPD), Dacryocystitis,dermatomyositis, Diabetic retinopathy, Diabetes mellitus, Diskherniation, Disk prolaps, Drug induced immune hemolytic anemia,Endocarditis, Endometriosis, endophthalmitis, Episcleritis, Erythemamultiforme, erythema multiforme major, Gestational pemphigoid,Guillain-Barré Syndrome (GBS), Hay Fever, Hughes Syndrome, IdiopathicParkinson's Disease, idiopathic interstitial pneumonia, IgE-mediatedAllergy, Immune hemolytic anemia, Inclusion Body Myositis, Infectiousocular inflammatory disease, Inflammatory demyelinating disease,Inflammatory heart disease, Inflammatory kidney disease, IPF/UIP,Iritis, Keratitis, Keratojuntivitis sicca, Kussmaul disease orKussmaul-Meier Disease, Landry's Paralysis, Langerhan's CellHistiocytosis, Livedo reticularis, Macular Degeneration, MicroscopicPolyangiitis, Morbus Bechterev, Motor Neuron Disorders, Mucous membranepemphigoid, Multiple Organ failure, Myasthenia Gravis, MyelodysplasticSyndrome, Myocarditis, Nerve Root Disorders, Neuropathy, Non-A Non-BHepatitis, Optic Neuritis, Osteolysis, Pauciarticular JRA, peripheralartery occlusive disease (PAOD), peripheral vascular disease (PVD),peripheral artery disease (PAD), Phlebitis, Polyarteritis nodosa (orperiarteritis nodosa), Polychondritis, Polymyalgia Rheumatica, Poliosis,Polyarticular JRA, Polyendocrine Deficiency Syndrome, Polymyositis,polymyalgia rheumatica (PMR), Post-Pump Syndrome, primary parkinsonism,Prostatitis, Pure red cell aplasia, Primary Adrenal Insufficiency,Recurrent Neuromyelitis Optica, Restenosis, Rheumatic heart disease,SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis),Scleroderma, Secondary Amyloidosis, Shock lung, Scleritis, Sciatica,Secondary Adrenal Insufficiency, Silicone associated connective tissuedisease, Sneddon-Wilkinson Dermatosis, spondilitis ankylosans,Stevens-Johnson Syndrome (SJS), Systemic inflammatory response syndrome,Temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis,Transverse myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1allergic reaction, Type II Diabetes, Urticaria, Usual interstitialpneumonia (UIP), Vasculitis, Vernal conjunctivitis, viral retinitis,Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet macular degeneration,and Wound healing.

In another aspect, the binding proteins of the invention are useful fortreating a disorder selected from the group consisting of AcuteLymphoblastic Leukemia, Acute Myeloid Leukemia, AdrenocorticalCarcinoma, Anal Cancer, Appendix Cancer, Cerebellar Astrocytoma,Cerebral Astrocytoma, Basal Cell Carcinoma, Bile Duct Cancer,Extrahepatic, Bladder Cancer, Bone Cancer, Osteosarcoma/MalignantFibrous Histiocytoma Brain Stem Glioma, Brain Tumor, Brain Stem Glioma,Cerebral strocytoma/Malignant Glioma, Ependymoma, Medulloblastoma,Supratentorial Primitive Neuroectodermal Tumors, Visual Pathway andHypothalamic Glioma, Breast Cancer, Bronchial Adenomas/Carcinoids,Carcinoid Tumor, Carcinoid Tumor, Gastrointestinal Carcinoma of UnknownPrimary, Central Nervous System Lymphoma, Primary CerebellarAstrocytoma, Cervical Cancer, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia Chronic Myeloproliferative Disorders, Colon Cancer,Colorectal Cancer, Cutaneous T-Cell Lymphoma, Endometrial Cancer,Ependymoma, Esophageal Cancer, Ewing Family of Tumors, Extracranial GermCell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer,Eye Cancer, Intraocular Melanoma Retinoblastoma, Gallbladder Cancer,Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor,Gastrointestinal Stromal Tumor (GIST), Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Ovarian Germ Cell Tumor, GestationalTrophoblastic Tumor, Glioma, Brain Stem Glioma, Cerebral AstrocytomaGlioma, Childhood Visual Pathway and Hypothalamic Glioma, Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer, HodgkinLymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet CellCarcinoma (Endocrine Pancreas), Kaposi Sarcoma, Kidney (Renal Cell)Cancer, Laryngeal Cancer, Acute Lymphoblastic Leukemia, Acute MyeloidLeukemia, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia,Hairy Cell Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Non-SmallCell Lung Cancer, Small Cell Lung Cancer, AIDS-Related Lymphoma, BurkittLymphoma, Cutaneous T-Cell Lymphoma, Hodgkin Lymphoma, Non-HodgkinLymphoma, Primary Central Nervous System Lymphoma, WaldenströmMacroglobulinemia, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma,Medulloblastoma, Melanoma, Intraocular (Eye) Melanoma, Merkel CellCarcinoma, Malignant Mesothelioma, Metastatic Squamous Neck Cancer withOccult Primary, Mouth Cancer, Multiple Endocrine Neoplasia Syndrome,Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides,Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Diseases,Myelogenous Leukemia, Chronic Myeloid Leukemia, Multiple Myeloma,Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer,Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Oral Cavity Cancer,Lip and Oropharyngeal Cancer, Osteosarcoma/Malignant FibrousHistiocytoma of Bone, Ovarian Cancer, Ovarian Epithelial Cancer, OvarianGerm Cell Tumor, Ovarian Low Malignant Potential Tumor, PancreaticCancer, Islet Cell Pancreatic Cancer, Paranasal Sinus and Nasal CavityCancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer,Pheochromocytoma, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Pleuropulmonary Blastoma, Prostate Cancer, Rectal Cancer, RenalCell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer,Retinoblastoma, Salivary Gland Cancer, Sarcoma, Ewing Family of Tumors,Kaposi Sarcoma, Soft Tissue Sarcoma, Uterine Sarcoma, Sézary Syndrome,Skin Cancer (Nonmelanoma), Skin Cancer (Melanoma), Merkel Cell SkinCarcinoma, Small Intestine Cancer, Squamous Cell Carcinoma, MetastaticSquamous Neck Cancer with Occult Primary, Stomach (Gastric) Cancer,Supratentorial Primitive Neuroectodermal Tumors, Cutaneous T-CellLymphoma, Testicular Cancer, Throat Cancer, Thymoma, Thymoma and ThymicCarcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvisand Ureter, Gestational Trophoblastic Tumor, Ureter and Renal Pelvis,Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, EndometrialUterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenström Macroglobulinemia, Wilms Tumor.

In another aspect the invention provides a method of treating a patientsuffering from a disorder in which human IL-13 is detrimental comprisingthe step of administering any one of the binding proteins disclosedabove before, concurrent, or after the administration of a second agent,as discussed above. In a preferred embodiment the additional therapeuticagent that can be coadministered and/or coformulated with one or moreIL-13 antagonists, (e.g., anti-IL-13 antibodies or fragments thereof,)include, but are not limited to, one or more of: inhaled steroids; oralsteroids; beta-agonists, e.g., short-acting or long-actingbeta-agonists; antagonists of leukotrienes or leukotriene receptors;combination drugs such as ADVAIR; IgE inhibitors, e.g., anti-IgEantibodies (e.g., XOLAIR); phosphodiesterase inhibitors (e.g., PDE4inhibitors); xanthines; anticholinergic drugs; mast cell-stabilizingagents such as cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3inhibitors; antagonists of histamine or its receptors including H1, H2,H3, and H4, and antagonists of prostaglandin D or its receptors (DP1 andCRTH2). Such combinations can be used to treat asthma and otherrespiratory disorders. Additional examples of therapeutic agents thatcan be coadministered and/or coformulated with one or more anti-IL-13antibodies or fragments thereof include one or more of: TNF antagonists(e.g., a soluble fragment of a TNF receptor, e.g., p55 or p75 human TNFreceptor or derivatives thereof, e.g., 75 kD TNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL)); TNF enzyme antagonists, e.g., TNFconverting enzyme (TACE) inhibitors; muscarinic receptor antagonists;TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeuticagents, e.g., methotrexate, leflunomide, or a sirolimus (rapamycin) oran analog thereof, e.g., CCI-779; COX2 and cPLA2 inhibitors; NSAIDs;immunomodulators; p38 inhibitors, TPL-2, MK-2 and NFkB inhibitors, amongothers. Additional second agent is selected from the group consisting ofbudenoside, epidermal growth factor, corticosteroids, cyclosporin,sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine,metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine,balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptorantagonists, anti-IL-1β monoclonal antibodies, anti-IL-6 monoclonalantibodies, growth factors, elastase inhibitors, pyridinyl-imidazolecompounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7,IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF, antibodies ofCD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or theirligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolatemofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone,phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents,complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAPkinase inhibitors, IL-1P converting enzyme inhibitors, TNFα convertingenzyme inhibitors, T-cell signalling inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors, soluble p55TNF receptor, soluble p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R,antiinflammatory cytokines, IL-4, IL-10, IL-11, and TGFβ.

In a preferred embodiment the pharmaceutical compositions disclosedabove are administered to the subject by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal,and transdermal.

One aspect of the invention provides at least one IL-13 anti-idiotypeantibody to at least one IL-13 binding protein of the present invention.The anti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one complementarily determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework region, or; any portionthereof, that can be incorporated into a binding protein of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to human IL-13 binding proteins, particularlyanti-IL-13 antibodies, or antigen-binding portions thereof, that bindIL-13. Various aspects of the invention relate to antibodies andantibody fragments, and pharmaceutical compositions thereof, as well asnucleic acids, recombinant expression vectors and host cells for makingsuch antibodies and fragments. Methods of using the antibodies of theinvention to detect human IL-13, to inhibit human IL-13 activity, eitherin vitro or in vivo; and to regulate gene expression are alsoencompassed by the invention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “Polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

The terms “human IL-13” and “human IL-13 wild type” (abbreviated hereinas h IL-13, h IL-13 wt), as used herein, includes a human cytokine thatis secreted primarily by T helper 2 cells. The term includes a monomericprotein of 13 kDa polypeptide. The structure of human IL-13 is describedfurther in, for example, (Moy, Diblasio et al. 2001 J Mol Biol 310219-30). The term human IL-13 is intended to include recombinant humanIL-13 (rh IL-13), which can be prepared by standard recombinantexpression methods. Table 1 shows the amino acid sequence of humanIL-13, SEQ ID No. 1, which is known in the art.

TABLE 1 Sequence of human IL-13 Sequence Sequence Protein Identifier12345678901234567890123456789012 Human IL-13 SEQ ID NO.: 1MALLLTTVIALTCLGGFASPGPVPPSTALREL IEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSA GQFSSLHVRDTKIEVAQFVKDLLLHLKKLFRE GRFN

The term “human IL-13 variant” (abbreviated herein as h IL-13v), as usedherein, includes a variant of human IL-13 wherein amino acid residue 130of SEQ ID NO. 1 is changed from Arginine to Glutamine (R130Q).

“Biological activity” as used herein, refers to all inherent biologicalproperties of the cytokine. Biological properties of IL-13 include butare not limited to binding IL-13 receptor; (other examples includeimmunoglobulin isotype switching to IgE in human B cells and suppressinginflammatory cytokine production).

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Nonlimitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 andIgA2) or subclass.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hIL-13). It has been shown that the antigen-binding function ofan antibody can be performed by fragments of a full-length antibody.Such antibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Human IgG heavychain and light chain constant domain amino acid sequences are known inthe art and represented in Table 2.

TABLE 2 Sequence of human IgG heavy chain constantdomain and light chain constant domain Sequence Sequence ProteinIdentifier 12345678901234567890123456789012 Ig gamma-1  SEQ ID NO.: 2ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant regionFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig gamma-1 SEQ ID NO.: 3ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant regionFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS mutant LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVRNAKTKPREEQYNSTYRVVSVLYVL HQDMLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYILPPSREEMIKNQVSLICLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig Kappa constantSEQ ID NO.: 4 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY regionPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda SEQ ID NO.: 5 QPKAAPSVTLFPPSSEELQANKATLVCLISDFconstant region YPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hIL-13 is substantially free of antibodies that specifically bindantigens other than hIL-13). An isolated antibody that specificallybinds hIL-13 may, however, have cross-reactivity to other antigens, suchas IL-13 molecules from other species. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II C, below), antibodies isolated from a recombinant,combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech.15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem.35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al(2000) Immunology Today 21:364-370) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.One embodiment provides fully human antibodies capable of binding humanIL-13 which can be generated using techniques well known in the art,such as, but not limited to, using human Ig phage libraries such asthose disclosed in Jermutus et al., PCT publication No. WO 2005/007699A2.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences. In one embodiment, humanized anti human IL-13 antibodies andantigen binding portions are provided. Such antibodies were generated byobtaining murine anti-hIL-13 monoclonal antibodies using traditionalhybridoma technology followed by humanization using in vitro geneticengineering, such as those disclosed in Kasaian et al PCT publicationNo. WO 2005/123126 A2.

The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although preferred embodiments useKabat or Chothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention the human heavy chain and lightchain acceptor sequences are selected from the sequences described inTable 3 and Table 4.

TABLE 3 HEAVY CHAIN ACCEPTOR SEQUENCES Sequence SEQ ID No.Protein region 12345678901234567890123456789012 6 VH1-18&JH6 FR1QVQLVQSGAEVKKPGASVKVSCKASGYTFT 7 VH1-18&JH6 FR2 WVRQAPGQGLEWMG 8VH1-18&JH6 FR3 RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR 9 VH1-18&JH6 FR4WGQGTTVTVSS 6  21/28&JH4 FR1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 10 21/28&JH4 FR2 WVRQAPGQRLEWMG 11  21/28&JH4 FR3RVTITRDTSASTAYMELSSLRSEDTAVYYCAR 12  21/28&JH4 FR4 WGQGTLVTVSS 13VH2-26&JH6 FR1 QVTLKESGPVLVKPTETLTLTCTVSGFSLS 14 VH2-26&JH6 FR2WIRQPPGKALEWLAH 15 VH2-26&JH6 FR3 RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR 9VH2-26&JH6 FR4 WGQGTTVTVSS 16    M60&JH4 FR1QVTLRESGPALVKPTQTLTLTCTLYGFSLS 17    M60&JH4 FR2 WIRQPPGKALEWLA 18   M60&JH4 FR3 RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR 12    M60&JH4 FR4WGQGTLVTVSS 6 VH1-46&JH6 FR1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 7VH1-46&JH6 FR2 WVRQAPGQGLEWMG 19 VH1-46&JH6 FR3RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR 9 VH1-46&JH6 FR4 WGQGTTVTVSS

TABLE 4 LIGHT CHAIN ACCEPTOR SEQUENCES Sequence SEQ ID No.Protein region 12345678901234567890123456789012 20    A20&JK4 FR1DIQMTQSPSSLSASVGDRVTITC 21    A20&JK4 FR2 WYQQKPGKVPKLLIY 22   A20&JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC 23    A20&JK4 FR4FGGGTKVEIKR 20 III-3R&JK4 FR1 DIQMTQSPSSLSASVGDRVTITC 24 III-3R&JK4 FR2WYQQKPGKAPKLLIY 25 III-3R&JK4 FR3 GVPSRISGSGSGTDFTFTISSLQPEDIATYYC 23III-3R&JK4 FR4 FGGGTKVEIKR 26     A1&JK4 FR1 DVVMTQSPLSLPVTLGQPASISC 27    A1&JK4 FR2 WFQQRPGQSPRRLIY 28     A1&JK4 FR3GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 23     A1&JK4 FR4 FGGGTKVEIKR 29    01&JK2 FR1 DIVMTQTPLSLPVTPGEPASISC 30     01&JK2 FR2 WYLQKPGQSPQLLIY28     01&JK2 FR3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 31     01&JK2 FR4FGQGTKLEIKR

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med. Biol. 484:13-30(2001)). One of the advantages provided by various embodiments of thepresent invention stems from the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or fragment thereof which immunospecificallybinds to an antigen of interest and which comprises a framework (FR)region having substantially the amino acid sequence of a human antibodyand a complementary determining region (CDR) having substantially theamino acid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, preferably at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to the amino acidsequence of a non-human antibody CDR. A humanized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a human immunoglobulin consensus sequence. Preferably, ahumanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “multivalent binding protein” is used in this specification todenote a binding protein comprising two or more antigen binding sites.The multivalent binding protein is preferably engineered to have thethree or more antigen binding sites, and is generally not a naturallyoccurring antibody. The term “multispecific binding protein” refers to abinding protein capable of binding two or more related or unrelatedtargets. Dual variable domain (DVD) binding proteins as used herein, arebinding proteins that comprise two or more antigen binding sites and aretetravalent or multivalent binding proteins. Such DVDs may bemonospecific, i.e capable of binding one antigen or multispecific, i.e.capable of binding two or more antigens. DVD binding proteins comprisingtwo heavy chain DVD polypeptides and two light chain DVD polypeptidesare referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chainDVD polypeptide, and a light chain DVD polypeptide, and two antigenbinding sites. Each binding site comprises a heavy chain variable domainand a light chain variable domain with a total of 6 CDRs involved inantigen binding per antigen binding site.

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of a cytokine when a binding protein specificallybinds the cytokine. Preferably a neutralizing binding protein is aneutralizing antibody whose binding to hIL-13 and/or hIL-13 results ininhibition of a biological activity of hIL-13 and/or hIL-13. Preferablythe neutralizing binding protein binds hIL-13 and/or hIL-13 and reducesa biologically activity of IL-13 and/or hIL-13 by at least about 20%,40%, 60%, 80%, 85% or more Inhibition of a biological activity of hIL-13and/or hIL-13 by a neutralizing binding protein can be assessed bymeasuring one or more indicators of hIL-13 and/or hIL-13 biologicalactivity well known in the art. For example inhibition of human IL-13induced production of TARC(CCL-17) by A-549 cells (see Example 1.1.C).

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-hIL-13 antibody that binds to an IL-13 antigen and/or theneutralizing potency of an antibody, for example, an anti-hIL-13antibody whose binding to hIL-13 inhibits the biological activity ofhIL-13, e.g For example inhibition of human IL-13 induced production ofTARC(CCL-17) by A-549 cells (see Example 1.1.C).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIAcoresystem (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).For further descriptions, see Jönsson, U., et al. (1993) Ann. Biol.Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627;Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson,B., et al. (1991) Anal. Biochem. 198:268-277.

The term “k_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “k_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The term “labeled binding protein” as used herein, refers to a proteinwith a label incorporated that provides for the identification of thebinding protein. Preferably, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof.

The terms “crystal”, and “crystallized” as used herein, refer to anantibody, or antigen binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter, which isdistinct from other forms such as the amorphous solid state or theliquid crystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well-understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2ndea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).”

The term “polynucleotide” as referred to herein means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide”: is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Protein constructs of the presentinvention may be expressed, and purified using expression vectors andhost cells known in the art, including expression cassettes, vectors,recombinant host cells and methods for the recombinant expression andproteolytic processing of recombinant polyproteins and pre-proteins froma single open reading frame (e.g., WO 2007/014162 incorporated herein byreference).

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the Kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. Coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct, which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of hIL-13).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of hIL-13). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies are described, e.g., in WO01/83525.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist. Particular agonists of interest may include, but are notlimited to, IL-13 polypeptides or polypeptides, nucleic acids,carbohydrates, or any other molecules that bind to hIL-13.

The term “antagonist” or “inhibitor”, as used herein, refer to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological or immunologicalactivity of hIL-13 and/or hIL-13. Antagonists and inhibitors of hIL-13and/or hIL-13 may include, but are not limited to, proteins, nucleicacids, carbohydrates, or any other molecules, which bind to hIL-13and/or hIL-13.

The term “inhibit binding to the receptor” refers to the ability of thebinding protein to prevent the binding of IL-13 to one or more of itsreceptors. Such inhibition of binding to the receptor would result indiminishing or abolishing the biological activity mediated by binding ofIL-13 to its receptor or receptors.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, serum, urine, synovial fluid, cells,organs, tissues, bone marrow, lymph nodes and spleen.

I. Antibodies that Bind Human IL-13.

One aspect of the present invention provides isolated murine monoclonalantibodies, or antigen-binding portions thereof, that bind to IL-13 withhigh affinity, a slow off rate and high neutralizing capacity. A secondaspect of the invention provides chimeric antibodies that bind IL-13. Athird aspect of the invention provides humanized antibodies, orantigen-binding portions thereof, that bind IL-13. Preferably, theantibodies, or portions thereof, are isolated antibodies. Preferably,the antibodies of the invention are neutralizing human anti-IL-13 and/orhuman anti-IL-13 antibodies.

A. Method of Making Anti IL-13 Antibodies

Antibodies of the present invention may be made by any of a number oftechniques known in the art.

1. Anti-IL-13 Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention (See Example 1.2). Briefly, mice canbe immunized with an IL-13 antigen. In a preferred embodiment, the IL-13antigen is administered with an adjuvant to stimulate the immuneresponse. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks.

After immunization of an animal with an IL-13 antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-IL-13antibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-IL-13 antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen IL-13 are detected in the mouse serum, the mouse spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the ATCC. Hybridomas are selected andcloned by limited dilution. The hybridoma clones are then assayed bymethods known in the art for cells that secrete antibodies capable ofbinding IL-13. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using IL-13, or a portionthereof, or a cell expressing IL-13. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example ofELISA screening is provided in WO 00/37504, herein incorporated byreference.

Anti-IL-13 antibody-producing hybridomas are selected, cloned andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

In a preferred embodiment, the hybridomas are mouse hybridomas, asdescribed above. In another preferred embodiment, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-IL-13 antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

2. Anti-IL-13 Monoclonal Antibodies Using SLAM

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S.et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals described in Section 1, arescreened using an antigen-specific hemolytic plaque assay, wherein theantigen IL-13, a subunit of IL-13, or a fragment thereof, is coupled tosheep red blood cells using a linker, such as biotin, and used toidentify single cells that secrete antibodies with specificity forIL-13. Following identification of antibody-secreting cells of interest,heavy- and light-chain variable region cDNAs are rescued from the cellsby reverse transcriptase-PCR and these variable regions can then beexpressed, in the context of appropriate immunoglobulin constant regions(e.g., human constant regions), in mammalian host cells, such as COS orCHO cells. The host cells transfected with the amplified immunoglobulinsequences, derived from in vivo selected lymphocytes, can then undergofurther analysis and selection in vitro, for example by panning thetransfected cells to isolate cells expressing antibodies to IL-13. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation methods such as those describedin PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

3. Anti-IL-13 Monoclonal Antibodies Using Transgenic Animals

In another embodiment of the instant invention, antibodies are producedby immunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with an IL-13 antigen. In a preferred embodiment,the non-human animal is a XENOMOUSE transgenic mouse, an engineeredmouse strain that comprises large fragments of the human immunoglobulinloci and is deficient in mouse antibody production. See, e.g., Green etal. Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771,5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO 94/02602,published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both published Oct.31, 1996, WO 98/16654, published Apr. 23, 1998, WO 98/24893, publishedJun. 11, 1998, WO 98/50433, published Nov. 12, 1998, WO 99/45031,published Sep. 10, 1999, WO 99/53049, published Oct. 21, 1999, WO 0009560, published Feb. 24, 2000 and WO 00/037504, published Jun. 29,2000. The XENOMOUSE transgenic mouse produces an adult-like humanrepertoire of fully human antibodies, and generates antigen-specifichuman Mabs. The XENOMOUSE transgenic mouse contains approximately 80% ofthe human antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci and xlight chain loci. See Mendez et al., Nature Genetics 15:146-156 (1997),Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures ofwhich are hereby incorporated by reference.

4. Anti-IL-13 Monoclonal Antibodies Using Recombinant Antibody Libraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al.PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling etal. PCT Publication No. WO 93/01288; McCafferty et al. PCT PublicationNo. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchset al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum AntibodHybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCaffertyet al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson etal. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS88:7978-7982, US patent application publication 20030186374, and PCTPublication No. WO 97/29131, the contents of each of which areincorporated herein by reference.

The recombinant antibody library may be from a subject immunized withIL-13 or IL-13, or a portion of IL-13 or IL-13. Alternatively, therecombinant antibody library may be from a naïve subject, i.e., one whohas not been immunized with IL-13, such as a human antibody library froma human subject who has not been immunized with human IL-13. Antibodiesof the invention are selected by screening the recombinant antibodylibrary with the peptide comprising human IL-13 to thereby select thoseantibodies that recognize IL-13. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for hIL-13, such as thosethat dissociate from human IL-13 with a particular k_(off) rateconstant, the art-known method of surface plasmon resonance can be usedto select antibodies having the desired k_(off) rate constant. To selectantibodies of the invention having a particular neutralizing activityfor hIL-13, such as those with a particular an IC₅₀, standard methodsknown in the art for assessing the inhibition of hIL-13 activity may beused.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human IL-13. Preferably, theantibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of dualspecificity antibodies of the invention. One type of alternativeexpression system is one in which the recombinant antibody library isexpressed as RNA-protein fusions, as described in PCT Publication No. WO98/31700 by Szostak and Roberts, and in Roberts, R. W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, acovalent fusion is created between an mRNA and the peptide or proteinthat it encodes by in vitro translation of synthetic mRNAs that carrypuromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, aspecific mRNA can be enriched from a complex mixture of mRNAs (e.g., acombinatorial library) based on the properties of the encoded peptide orprotein, e.g., antibody, or portion thereof, such as binding of theantibody, or portion thereof, to the dual specificity antigen. Nucleicacid sequences encoding antibodies, or portions thereof, recovered fromscreening of such libraries can be expressed by recombinant means asdescribed above (e.g., in mammalian host cells) and, moreover, can besubjected to further affinity maturation by either additional rounds ofscreening of mRNA-peptide fusions in which mutations have beenintroduced into the originally selected sequence(s), or by other methodsfor affinity maturation in vitro of recombinant antibodies, as describedabove.

In another approach the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed Wittrup, etal. U.S. Pat. No. 6,699,658 incorporated herein by reference.

B. Production of Recombinant IL-13 Antibodies

Antibodies of the present invention may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the heavy and light chains is(are) transfected into a host cell by standard techniques. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

1. Anti IL-13 Antibodies

Table 5 is a list of amino acid sequences of VH and VL regions ofpreferred anti-hIL-13 antibodies of the invention.

TABLE 5 List of Amino Acid Sequences of VH and VL regions SEQ IDSequence No. Protein region 123456789012345678901234567890 32 VH 25C8QVQLQQPGAELVRPGASVQLSCKASGYTFT SSWIHWVNQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSED SAVYYCASTATDFDYWGQGTTLTVSS VH 25C8 CDR-H1Residues 31-35 SSWIH of SEQ ID NO.: 32 VH 25C8 CDR-H2 Residues 50-66MIRPSDSETKLNQKFKD of SEQ ID NO.: 32 VH 25C8 CDR-H3 Residues 99- TATDFDY105 of SEQ ID NO.: 32 33 VL 25C8 DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRF SGAPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQHNYLPLTFGAGTNLELKR VL 25C8 CDR-L1 Residues 24-39 KSTKSLLNSDGFTYLDof SEQ ID NO.: 33 VL 25C8 CDR-L2 Residues 55-61 LVSNRFS of SEQ IDNO.: 33 VL 25C8 CDR-L3 Residues 94-102 FQHNYLPLT of SEQ ID NO.: 33 34VH 9C11 QVRLQQPGAELVRPGASVKLSCKASGYTFT SSWIHWVNQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSED SAVYYCASTATDFDYWGQGTTLTVSS VH 9C11 CDR-H1Residues 31-35 SSWIH of SEQ ID NO.: 34 VH 9C11 CDR-H2 Residues 50-66MIHPSDSETRLANKFKD of SEQ ID NO.: 34 VH 9C11 CDR-H3 Residues 99- TATDFDY105 of SEQ ID NO.: 34 35 VL 9C11 DVVLTQTPLSLPVNIGDQASISCRSTQTLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQNNYLPLTFGAGTKLELKR VL 9C11 CDR-L1 Residues24-39 RSTQTLLNSDGFTYLDof SEQ ID NO.: 35 VL 9C11 CDR-L2 Residues 55-61 LVSNRFS of SEQ IDNO.: 35 VL 9C11 CDR-L3 Residues 94-102 FQNNYLPLT of SEQ ID NO.: 35 36VH 21D9 QVQLQQSGDDLVKPGASVKLSCKASGYTFT SYWINWIKQRPGQGLEWIGHIAPGSGETYDNEMEKDKAKLTVDTSSNTAYIHLSSLSSED SAVYFCARGSFTFFYAMDYWGQGTSVTVSSVH 21D09 CDR-H1 Residues 31-35 SYWIN SEQ ID NO.: 36 VH 21D9 CDR-H2Residues 50-66 HIAPGSGETYDNEMFKD of SEQ ID NO.: 36 VH 21D9 CDR-H3Residues 99- GSFTFFYAMDY 109 of SEQ ID NO.: 36 37 VL 21D9DVLMTQTPLSLPVSLGDQASISCRSSQNIV HSNGKTYLEWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV YYCFQGSHVPYTFGGGTKLEIKR VL 21D9 CDR-L1Residues 24-39 RSSQNIVHSNGKTYLE of SEQ ID NO.: 37 VL 21D9 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO.: 37 VL 21D9 CDR-L3 Residues 94-102FQGSHVPYT of SEQ ID NO.: 37 38 VH 22D10 QVQLQQSGDDLVKPGASVKLSCKASGYTFTSYWINWIKQRPGQGLEWIGHIAPGSGETYD NEMFKDKAKLTVOTSSSTAYIHLSSLSSEDSAVYFCARGSFTFFYAMDYWGQGTSVTVSS VH 22D10 CDR-H1 Residues 1-35 SYWINof SEQ ID NO.: 38 VH 22D10 CDR-H2 Residues 50-66 HIAPGSGETYDNEMFKDof SEQ ID NO.: 38 VH 22010 CDR-H3 Residues 99- GSFTFFYAMDY 109 of SEQ IDNO.: 38 37 VL 22D10 DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGKTYLEWYLQRPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKR VL 22D10 CDR-L1 Residues 24-39 RSSQNIVHSNGKTYLEof SEQ ID NO.: 37 VL 22D10 CDR-L2 Residues 55-61 KVSNRFS of SEQ IDNO.: 37 VL 22D10 CDR-L3 Residues 94-102 FQGSHVPYT of SEQ ID NO.: 37 39VH 5F1 QVQLQQSGAELARPGTSVKLSCKASGYTFT TYGISWVKQRTGQGLEWIGEIYPGSYNTYYNEKFRGKATLTADKSSSTAYMQLSSLTSED SAVYFCSRWRTSYFSDYGYFDYWGQGTTLT VSSVH 5F1 CDR-H1 Residues 31-35 TYGIS of SEQ ID NO.: 39 VH 5F1 CDR H2Residues 50-66 EIYPGSYNTYYNEKFRG of SEQ ID NO.: 39 VH 5F1 CDR-H3Residues 99- WRTSYFSDYGYFDY 112 of SEQ ID NO.: 39 40 VL 5F1DVVMTQTPDSDPVSDGDQASISCRSSQSLV HSHGNTYLHWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV YFCSQSTHVPYTFGGGTKLEIKR VL 5F1 CDR-L1Residues 24-39 of SEQ ID RSSQSLVHSHGNTYLH NO.: 40 VL 5F1 CDR-L2Residues 55-61 TVSNRFS of SEQ ID NO.: 40 VL 5F1 CDR-L3 Residues 94-102SQSTHVPYT of SEQ ID NO.: 40 41 VH 5G1 QVQLQQSGAELARPGTSVKLSCKASGYTFTTYGVSWVKQRTGQGLEWIGEIYPGNYNTYY NEKFRGKATDTADKSSSTAYMQDSSDTSEDSAVYFCSRWRTSYFSDYGYFDYWGQGTTLT VSS VH 5G1 CDR-H1 Residues 31-35 TYGVSof SEQ ID NO.: 41 VH 5G1 CDR-H2 Residues 50-66 EIYPGNYNTYYNEKFRGof SEQ ID NO.: 41 VH 5G1 D1-H3 Residues 99- WRTSYFSDYYFDY 112 of SEQ IDNO.: 41 40 VL 5G1 DVVMTQTPDSDPVSDGDQASISCRSSQSLVHSHGNTYLHWYLQKPGQSPKLLIYTVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKR VL 5G1 CDR-L1 Residues 24-39 RSSQSLVHSHGNTYLHof SEQ ID NO.: 40 VL 5G1 CDR-L2 Residues 55-61 TVSNRFS of SEQ ID NO.: 40VL 5G1 CDR-L3 Residues 94-102 SQSTHVPYT of SEQ ID NO.: 40 42 VH 3H7EVQDVESGGGLVKPGGSLKLSCAASGFTFS TYAMSWVRQTPEKRLFWVAGISSGGSYTYYPETMKGRFTISRDNARNTLYLQMSSLRSED TAIYYCTRGSWGQGTSVTVSS VH 3H7 CDR-H1Residues 31-35 TYAMS of SEQ ID NO.: 42 VH 3H7 CDR-H2 Residues 50-66GISSGGSYTYYPETMKG of SEQ ID NO.: 42 VH 3H7 CDR-H3 Residues 99- GSof SEQ ID NO.: 42 43 VL 3H7 DVVLTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLLQRPGQSPKRLIYLVSKLD SGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKR VL 3H7 CDR-L1 Residues 24-39 KSSQSLLDSDGETYLNof SEQ ID NO.: 43 VL 3H7 CDR-L2 Residues 55-61 LVSKLDS of SEQ ID NO.: 43VL 3H7 CDR-L3 Residues 94-102 WQGTHFPWT of SEQ ID NO.: 43 44 VH 14B2EVKLVESGGGLVRPGGSLKLSCAASGFTES SYAMNWVRQTPEKRLEWVASISSGGNIYYSDSVKGRFTISRDNARNTLHLQMSSLRSEDT AMYYCARDGYLYAMDYWGQGTSVTVSSVH 14B2 CDR-H1 Residues 31-35 SYAMN of SEQ ID NO.: 44 VH 14B2 CDR-H2Residues 50-65 SISSGGNIYYSDSVKG of SEQ ID NO.: 44 VH 14B2 CDR-H3Residues 98- DGYLYAMDY 106 of SEQ ID NO.: 44 45 VL 14B2DIVMSQSPSSLAVSVGEKVTMSCKSSQNLL YSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLA VYYCQQYYSYPFTFGSGTKLEIKR VL 14B2 CDR-L1Residues 24-40 KSSQNLLYSSNQKNYLA of SEQ ID NO.: 45 VL 14B2 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 45 VL 14B2 CDR-L3 Residues 95-103QQYYSYPFT of SEQ ID NO.: 45 46 VH 13C5 QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSDMGVDWIRQPSGKGLEWLAHIWWDDVKR YNPALKSRLTISKDTSSSQVFLMLASVDTADTATYYCARTVSSGYIYYAMDYWGQGTSVT VSS VH 13C5 CDR-H1 Residues 31-37 TSDMGVDof SEQ ID NO.: 46 VH 13C5 CDR-H2 Residues 52-67 HIWWDDVKRYNPALKSof SEQ ID NO.: 46 VH 13C5 CDR-H3 Residues 100- TVSSGYIYYAMIDY112 of SEQ ID NO.: 46 47 VL 13C5 DIQMTQTASSLSASLGDRVTISCRASQDIRNYLNWYQRKPDGTVKLLIFYTSKLHSGVPS RFSGSGSGTDYSLTIRNLEQEDIATYFCQQGNTLPLTFGGGTKLEIKR VL 1305 CDR-L1 Residues 24-34 RASQDIRNYLN of SEQ IDNO.: 47 VL 13C5 CDR-L2 Residues 50-56 YTSKLHS of SEQ ID NO.: 47VL 13C5 CDR-L3 Residues 89-97 QQGNTLPLT of SEQ ID NO.: 47 48 VH 29G5QVTLKESGPGILQRSQTLSLTCSFSGFSLS TSDMGVDWIRQPSGKDLEWLAHIWNDDVKRYNPALKSRLTISKDTSSSQVFLMLASVDTA DTATYYCARIVSSGYIYYALDYWGQGTSVT VSSVH 29G5 CDR-H1 Residues 31-37 TSDMGVD of SEQ ID NO.: 48 VH 29G5 CDR-H2Residues 52 67 HIWWDDVKRYNPALKS of SEQ ID NO.: 48 VH 29G5 CDR-H3Residues 100- IVSSGYIYYALDY 112 of SEQ ID NO.: 48 49 VL 29G5DIQMTQTASSLSASLGDRVTISCRASQDIR NYLNWYQRKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPLTFGGGTKLEIKR VL 29G5 CDR-L1Residues 24-34 RASQDIRNYLN of SEQ ID NO.: 49 VL 29G5 CDR-L2Residues 50-56 YTSRLHS of SEQ ID NO.: 49 VL 29G5 CDR-L3 Residues 89-97QQGNTLPLT of SEQ ID NO.: 49 50 VR 33C3 QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSDLGVGWIRQPSGKGLEWLAHIWWDDVKR YNPALKSRLTISKDTDSSQVFLMIASVDTADTATYYCARIGSSGYIYYEMDYWGQGTSVT VSS VH 33C3 CDR-H1 Residues 31-37 TSDLGVGof SEQ ID NO.: 50 VH 33C3 CDR-H2 Residues 52-67 HIWWDDVICRYNPALKSof SEQ ID NO.: 50 VH 33C3 CDR-H3 Residues 100- IGSSGYIYYEMDY112 of SEQ ID NO.: 50 51 VL 33C3 DIQMTQTTSSLSASLGDRVTITCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPS RFSGSGSGTDYSLTISNLDQEDIATYFCQQGNTLPLTFGGGTRLEIKR VL 33C3 CDR-L1 Residues 24-34 RASQDIRNYLN of SEQ IDNO.: 51 VL 33C3 CDR-L2 Residues 50-56 YTSRLHS of SEQ ID NO.: 51VL 33C3 CDR-L3 Residues 89-97 QQGNTLPLT of SEQ ID NO.: 51 52 VH 4A8EVQLQQSGAEFVRFGALVKLSCKASGFNIK DYYMYWVKQRPEQGLEWIGRIDPENGNTIYDPKFQGKASITGDTSSNTAYLQLSSLTSED TAVYYCARYAYYGPFDYWGQGTTLTVSSVH 4A8 CDR-H1 Residues 31-35 DYYMY of SEQ ID NO.: 52 VH 4A8 CDR-H2Residues 50-66 RIDPENGNTIYDPKFQG of SEQ ID NO.: 52 VH 4A8 CDR-H3 Residues 99- YAYYGPFDY 107 of SEQ ID NO.: 52 53 VL 4A8QAVVTQESALTTSFGETVTLTCRSSIGTVT TNNYANWVQEKPDHLFTGLIGSTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHWVFGGGTKLTVLG VL 4A8 CDR-L1Residues 23-36 RSSIGTVTTNNYAN of SEQ ID NO.: 53 VL 4A8 CDR-L2Residues 52-58 STNNRAP of SEQ ID NO.: 53 VL 4A8 CDR-L3 Residues 9l-99ALWYSNHWV of SEQ ID NO.: 53 54 VH 1B6 QVQLKESGPGLVAPSQSLSITCTVSGESLTGYGVNWVRQPPGRGLEWLGMIWGDERIDYN SALKSRLSITKDNSKSQVFLKMNSLQTDDTGRYFCARDGYFPYAMDYWGQGTSVTVSS VH 1B6 CDR-H1 Residues 31-35 GYGVNof SEQ ID NO.: 54 VB 1B6 CDR-H2 Residues 50-65 MIWGDERIDYNSALKSof SEQ ID NO.: 54 VH 1B6 CDR-H3 Residues 98- DGYFPYAMDY 107 of SEQ IDNO.: 54 55 VL 1B6 NIVLTQSPASLAVSLGQRATISCRASETVDSYGKSYLHWYQQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLIIDPVEADDAATYYCQQNNEGPRTFGGGTKLEIKR VL 1B6 CDR-L1 Residues 24-38 RASETVDSYGKSYLHof SEQ ID NO.: 55 VL 1B6 CDR-L2 Residues 54-60 LASNLES of SEQ ID NO.: 55VL 1B6 CDR-L3 Residues 93-101 QQNNEGPRT of SEQ ID NO.: 55 56 VH 3E5QVQLKESGPGLVAPSQSLSITCTVSGFSLT GSSINWVRQPPGKGLEWLGMIWGDGRIDYNSVLKSRLSISKDSSKSQVFLKMNSLQADDT ARYYCARDGYYPYAMVYWGSNTSVTVSSVH 3E5 CDR-H1 Residues 31-35 GSSIN of SEQ ID NO.: 56 VH 3E5 CDR-H2Residues 50-65 MIWGDGRIDYNSVLKS of SEQ ID NO.: 56 VH 3E5 CDR-H3Residues 98- DGYYPYAMVY 107 of SEQ ID NO.: 56 57 VL 3E5NIVLTQSPASLAVSLGQRATIFCRASESVD SYGNSFMHWYQQKSGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATF YCQQNNENPRTFGGGTKLEIKR VL 3E5 CDR-L1Residues 24-38 RASESVDSYGNSFMH of SEQ ID NO.: 57 VL 3E5 CDR-L2Residues 54-60 LASNLES of SEQ ID NO.: 57 VL 3E5 CDR-L3 Residues 93-101QQNNENPRT of SEQ ID NO.: 57 58 VH 6C8 QVQLKESGPGLVAPSQSLSITCTVSEFSLTGSSVNWVRQPPGKGLEWLGMIWGDGRIDYN SALKSRLSIDKDNSKSQVFLKMNSLQTDDTARYYCARDGYYPYAMNYWGSNTSVTVSS VH 6C8 CDR-H1 Residues 31-35 GSSVNof SEQ ID NO.: 58 VH 6C8 CDR-H2 Residues 50-65 MIWGDGRIDYNSALKSof SEQ ID NO.: 58 VH 6C8 CDR-H3 Residues 98- DGYYPYAMNY 107 of SEQ IDNo.: 58 59 VL 6C8 NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYLASNLES GVPARFSGSGSRADFTLTIDPVEADDAATYYCQQNNENPRTFGGGTKLEIKR VL 6C8 CDR-L1 Residues 24-38 RASESVDSYGNSFMHof SEQ ID NO.: 59 VL 6C8 CDR-L2 Residues 54-60 LASNLES of SEQ ID NO.: 59VL 6C8 CDR-L3 Residues 93-101 QQNNENPRT of SEQ ID NO.: 59 60 VH 5D3QVQLKESGPGLVARSQSLSITCTVSGFSLT GYNINWVRQPPGKGLEWLGLIWGDGNTAFNSALKSRLSISKDNSKSQVFLKLNSLQTDDT ARYYCARDGYYPYAIKYWGQGTSVTVSSVH 5D3 CDR H1 Residues 31-35 GYNIN of SEQ ID NO.: 60 VH 5D3 CDR-H2Residues 50-65 LIWGDGNTAFNSALKS of SEQ ID NO.: 60 VH 5D3 CDR-H3Residues 98- DGYYPYAIKY 107 of SEQ ID NO.: 60 61 VL 5D3NIVLTQSPASLAVSLGQRATISCRASETVD SYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATY YCQQNNEDPRTFGGGTKLEIKR VL 5D3 CDR-L1Residues 24-38 RASETVDSYGNSFMH of SEQ ID NO.: 61 VL 5D3 CDR-L2Residues 54-60 LASNLES of SEQ ID NO.: 61 VL 5D3 CDR-L3 Residues 93-101QQNNEDPRT of SEQ ID NO.: 61 62 VH 8B6 QVQLKESGPGLVAPSQSLSITSTVSGESLTGHNINWVRQPPGKGLEWLGMIWGDGNTDFN SALKSRLSISKDNSKSQVFLKLNSLQTDDTARYYCARDGYYPYAIKFWGQGTSVTVSS VH 8B6 CDR-H1 Residues 31-35 GHNINof SEQ ID NO.: 62 VH 8B6 CDR-H2 Residues 50-65 MIWGDGNTDFNSALKSof SEQ ID NO.: 62 VH 8B6 CDR-H3 Residues 98- DGYYPYAIKF 107 of SEQ IDNO.: 62 63 VL 8B6 HIVLTQSPASLAVSLGQRATISCRASETVDSYGSSFLHWYQQKPGQPPKLLIYLASKLES GVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEGPRTFGGGSKLEIKR VL 8B6 CDR-L1 Residues 24-38 RASETVDSYGSSFLHof SEQ ID NO.: 63 VL 8B6 CDR-L2 Residues 54-60 LASKLES of SEQ ID NO.: 63VL 8B6 CDR-L3 Residues 93-101 QQNNEGPRT of SEQ ID NO.: 63

The foregoing isolated anti-IL-13 antibody CDR sequences establish anovel family of IL-13 binding proteins, isolated in accordance with thisinvention, and comprising polypeptides that include the CDR sequenceslisted in Table 6 below. To generate and to select CDR's of theinvention having preferred IL-13 binding and/or neutralizing activitywith respect to hIL-13 and or hIL-13, standard methods known in the artfor generating binding proteins of the present invention and assessingthe IL-13 and or IL-13 binding and/or neutralizing characteristics ofthose binding protein may be used, including but not limited to thosespecifically described herein.

TABLE 6 Consensus IL-13 CDR affinity ligands Sequence ConsensusCDR region Identifier Sequence CDR-H1 SEQ ID X₁     X₂ X₃ X₄ X₅ X₆ X₇NO.: 64 T        S D M G V D D          S W I H G         Y Y M SS             L A Y              H S N            N G CDR-H2 SEQ IDX₁         X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇NO.: 65 M                                I H P S D S E T R L N Q K F K DE                                — Y S G G Y N I Y Y P E M L R GH                                 A W E S G Y K I D S D S V Q SR                              D  G D E V D F D P T M S               SN R S A G                                W V L CDR-H3 SEQ IDX₁              X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ NO.: 66 W RT S Y F S D Y G Y F D Y T A F T F Y Y L A M V F G S Y Y G I Y P S N Y GS F P E L K D V I I CDR-L1 SEQ ID X₁              X₂ X₃ X₄  X₅ X₆ X₇ X₈X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ NO.: 67K                                      S S Q N L L Y S S N Q K N Y L AR                                       A T K S T Q N I D G F T F A D IT S V H T N N S M E G D H E H E T Y S N CDR-L2 SEQ ID X₁       X₂ X₃ X₄X₅ X₆ X₇ NO.: 68 L V S N R F S S T N K L D P K A T K E R T R H W M A Y PCDR-L3 SEQ ID X₁         X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ NO.: 69 F Q H N Y L P LT W                 L G S T V H F V Q Y T S F Y A W Y E Y W N L H N R GD P (alternative residues are listed below each amino acid position; —indicates residue may be absent).2. Anti IL-13 Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies are known in the art and discussed in detail in Example 2.1.See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202;U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which areincorporated herein by reference in their entireties. In addition,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger etal., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454which are incorporated herein by reference in their entireties) bysplicing genes from a mouse antibody molecule of appropriate antigenspecificity together with genes from a human antibody molecule ofappropriate biological activity can be used.

In one embodiment, the chimeric antibodies of the invention are producedby replacing the heavy chain constant region of the murine monoclonalanti human IL-13 antibodies described in section 1 with a human IgG1constant region. In a specific embodiment the chimeric antibody of theinvention comprises a heavy chain variable region (V_(H)) comprising theamino acid sequence of SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 41; SEQID NO: 42; SEQ ID NO: 46 and a light chain variable region (V_(L))comprising the amino acid sequence of SEQ ID NO: 35; SEQ ID NO: 37; SEQID NO: 40; SEQ ID NO: 43; or SEQ ID NO: 47.

3. Anti IL-13 Humanized Antibodies

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH-05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html; wwwhhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immuno-logy.html.www.immunologylink.com/;pathbox.wustl.edu/.about.hcenter/index.-html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html;www.biotech.ufl.edu/.about.fccl/protocol.html;www.isac-net.org/sites_geo.html; aximtlimt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nl/.about.jraats/linksl.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizhch/.about.honegger/AHOsem-inar/Slide01.html;www.cryst.bbk.ac.uk/.about.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html;www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

C. Production of Antibodies and Antibody-Producing Cell Lines

Preferrably, anti-IL-13 antibodies of the present invention, exhibit ahigh capacity to reduce or to neutralize IL-13 activity, e.g., asassessed by any one of several in vitro and in vivo assays known in theart (e.g., see Example 1.1.C). For example, these antibodies neutralizeIL-13-induced production of TARC by A-549 cells with IC₅₀ values in therange of at least about 10⁻⁸ M, about 10⁻⁹ M, or about 10⁻¹⁰ M.

In preferred embodiments, the isolated antibody, or antigen-bindingportion thereof, binds human IL-13, wherein the antibody, orantigen-binding portion thereof, dissociates from human IL-13 with ak_(off) rate constant of about 0.1s⁻¹ or less, as determined by surfaceplasmon resonance, or which inhibits human IL-13 and/or human IL-13activity with an IC₅₀ of about 1×10⁻⁶M or less. Alternatively, theantibody, or an antigen-binding portion thereof, may dissociate fromhuman IL-13 with a k_(off) rate constant of about 1×10⁻²s⁻¹ or less, asdetermined by surface plasmon resonance, or may inhibit human IL-13and/or human IL-13 activity with an IC₅₀ of about 1×10⁻⁷M or less.Alternatively, the antibody, or an antigen-binding portion thereof, maydissociate from human IL-13 with a k_(off) rate constant of about1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance, or mayinhibit human IL-13 and/or human IL-13 with an IC₅₀ of about 1×10⁻⁸ M orless. Alternatively, the antibody, or an antigen-binding portionthereof, may dissociate from human IL-13 with a k_(off) rate constant ofabout 1×10⁻⁴s⁻¹ or less, as determined by surface plasmon resonance, ormay inhibit IL-13 and/or human IL-13 activity with an IC₅₀ of about1×10⁻⁹M or less. Alternatively, the antibody, or an antigen-bindingportion thereof, may dissociate from human IL-13 with a k_(off) rateconstant of about 1×10⁻⁵s⁻¹ or less, as determined by surface plasmonresonance, or may inhibit IL-13 and/or human IL-13 activity with an IC₅₀of about 1×10⁻¹⁰M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human IL-13 with ak_(off) rate constant of about 1×10⁻⁵s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit IL-13 and/or human IL-13activity with an IC₅₀ of about 1×10⁻¹¹M or less.

IL-13 exerts its actions by binding to the IL-13 receptor (IL-13R) onthe cell surface, the heterodimer comprised of the IL-13Rα1 chain(IL-13Rα1) and the IL-4R chain (IL-4R). IL-13 binds to IL-13Rα1 firstwith low affinity (K_(D)=2-10 nM) and then recruits IL-4R to thecomplex, generating a high affinity receptor (K_(D)=0.03-0.4 nM) (Aman,M. J., et al. 1996 J. Biol. Chem. 271, 29265-29270; Miloux, et al. 1997FEBS Lett. 401, 163-166; Andrews, et al 2002 J. Biol. Chem. 277,46073-46078). Heterodimerization of IL-13R causes activation of Januskinases, TYK2 and JAK1, constitutively associated with IL-13Rα1 andIL-4R, respectively, followed by activation of the signal transducer andactivator of transcription 6 (STAT6) (Izuhara, K., and Arima, K. 2004Drug News Perspect. 17, 91-98). There is another IL-13-binding unit, theIL-13Rα2 chain (IL-13Rα2), which binds to IL-13 with high affinity(0.25-1.2 nM) (Caput, et al 1996J. Biol. Chem. 271, 16921-16926;Donaldson et al 1998 J. Immunol. 161, 2317-2324). No other receptormolecule is known to be involved in the IL-13•IL-13R2 complex. IL-13R2is initially thought to act as a nonsignaling “decoy” receptor. However,it was later discovered that it can bind to IL-13 and signals throughAP-1 pathway, leading to TNF-beta production in certain cell typesincluding macrophages, which in tern leads to lung fibrosis(Fichtner-Feigl, 2006 Nat Med 12:99-106). Therefore both IL-13Rα1/IL-4Rαand IL-13Rα2 pathways contribute to the overall pathophysiology ofasthma and other pulmonary inflammatory conditions. Therefore, atherapeutic anti-IL-13 antibody that blocks IL-13 binding to bothreceptors will be more effective that those that blocks only onereceptor.

We have isolated monoclonal antibodies that block IL-13 binding to bothIL-13αR1 and IL-13Rα2. Both ELISA-based receptor binding assay and125-I-labeled IL-13 binding assay on cell surface demonstrated that13C5, both murine version and humanized version (i.e. 13C5.5), were ableto effective block IL-13 binding to both receptors. Antibodies in thesame lineage as 13C5, including 25C8 and 33C3, were also able to blockIL-13 binding to both receptors. Epitope mapping of 13C5 indicated thatits binding site(s) included the C-terminal Helix D region of humanIL-13 (residues VRDTK IEVAQ FVKDL LLHLK KLFRE GR, corresponding to aminoacid 104-130 of SEQ ID NO. 1). The c-terminal helix D region has beenproposed to be involved in interactions with the IL-13 receptor (Zuegget al 2001 Immunol Cell Biol. 79:332-9). Crystal structure of humanIL-13 complexed with the Fab portion of 13C5.5 antibody indicated that13C5.5 binds the C-terminal Helix D region as well as the N-terminalHelix A region of human IL-13. Preferably the antibody, or antigenbinding fragment thereof binds human IL-13 such that IL-13 with saidantibody, or antigen binding fragment thereof, bound to the epitopedefined by the topographic regionsSer26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38and Lys123-Lys124-Leu125-Phe126-Arg127-Glu-128-Gly129-Arg130 of SEQ IDNo. 1 is inhibited from binding to the IL-13 receptor. Preferably theantibody, or antigen binding fragment thereof binds human IL-13 suchthat IL-13 with said antibody, or antigen binding fragment thereof,bound to the epitope defined by the topographic regionsArg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38 andLys123-Lys124-Leu125-Phe126-Arg127 of SEQ ID No. 1 is inhibited frombinding to the IL-13α2 receptor.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgG1 heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (Winter, et al. U.S. Pat. Nos.5,648,260; 5,624,821). The Fc portion of an antibody mediates severalimportant effector functions e.g. cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered.

One embodiment provides a labeled binding protein wherein an antibody orantibody portion of the invention is derivatized or linked to anotherfunctional molecule (e.g., another peptide or protein). For example, alabeled binding protein of the invention can be derived by functionallylinking an antibody or antibody portion of the invention (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

Another embodiment of the invention provides a crystallized bindingprotein. Preferably the invention relates to crystals of wholeanti-IL-13 antibodies and fragments thereof as disclosed herein, andformulations and compositions comprising such crystals. In oneembodiment the crystallized binding protein has a greater half-life invivo than the soluble counterpart of the binding protein. In anotherembodiment the binding protein retains biological activity aftercrystallization.

Crystallized binding protein of the invention may be produced accordingmethods known in the art and as disclosed in WO 02072636, incorporatedherein by reference.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Antibodies are glycoproteinswith one or more carbohydrate residues in the Fc domain, as well as thevariable domain. Carbohydrate residues in the Fc domain have importanteffect on the effector function of the Fc domain, with minimal effect onantigen binding or half-life of the antibody (R. Jefferis, Biotechnol.Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variabledomain may have an effect on the antigen binding activity of theantibody. Glycosylation in the variable domain may have a negativeeffect on antibody binding affinity, likely due to steric hindrance (Co,M. S., et al., Mol. Immunol. (1993) 30:1361-1367), or result inincreased affinity for the antigen (Wallick, S. C., et al., Exp. Med.(1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies.Glycosylation site mutants that retain the biological activity, but haveincreased or decreased binding activity, are another object of thepresent invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified antibody of the invention canbe made that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (eg.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S patent applications20040018590 and 20020137134 and PCT publication WO2005100584 A2).

In addition to the binding proteins, the present invention is alsodirected to an anti-idiotypic (anti-Id) antibody specific for suchbinding proteins of the invention. An anti-Id antibody is an antibody,which recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the binding protein or a CDR containingregion thereof. The immunized animal will recognize, and respond to theidiotypic determinants of the immunizing antibody and produce an anti-Idantibody. The anti-Id antibody may also be used as an “immunogen” toinduce an immune response in yet another animal, producing a so-calledanti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

D. Uses of Anti-IL-13 Antibodies

Given their ability to bind to human IL-13, the anti-human IL-13antibodies, or portions thereof, of the invention can be used to detecthuman IL-13 (e.g., in a biological sample, such as serum or plasma),using a conventional immunoassay, such as an enzyme linked immunosorbentassays (ELISA), an radioimmunoassay (RIA) or tissueimmunohistochemistry. The invention provides a method for detectinghuman IL-13 in a biological sample comprising contacting a biologicalsample with an antibody, or antibody portion, of the invention anddetecting either the antibody (or antibody portion) bound to human IL-13or unbound antibody (or antibody portion), to thereby detect human IL-13in the biological sample. The antibody is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ³H, ¹⁴C,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Alternative to labeling the antibody, human IL-13 can be assayed inbiological fluids by a competition immunoassay utilizing rhIL-13standards labeled with a detectable substance and an unlabeledanti-human IL-13 antibody. In this assay, the biological sample, thelabeled rhIL-13 standards and the anti-human IL-13 antibody are combinedand the amount of labeled rhIL-13 standard bound to the unlabeledantibody is determined. The amount of human IL-13 in the biologicalsample is inversely proportional to the amount of labeled rhIL-13standard bound to the anti-IL-13 antibody. Similarly, human IL-13 canalso be assayed in biological fluids by a competition immunoassayutilizing rhIL-13 standards labeled with a detectable substance and anunlabeled anti-human IL-13 antibody.

The antibodies and antibody portions of the invention preferably arecapable of neutralizing human IL-13 activity both in vitro and in vivo.Accordingly, such antibodies and antibody portions of the invention canbe used to inhibit hIL-13 activity, e.g., in a cell culture containinghIL-13, in human subjects or in other mammalian subjects having IL-13with which an antibody of the invention cross-reacts. In one embodiment,the invention provides a method for inhibiting hIL-13 activitycomprising contacting hIL-13 with an antibody or antibody portion of theinvention such that hIL-13 activity is inhibited. For example, in a cellculture containing, or suspected of containing hIL-13, an antibody orantibody portion of the invention can be added to the culture medium toinhibit hIL-13 activity in the culture.

In another embodiment, the invention provides a method for reducinghIL-13 activity in a subject, advantageously from a subject sufferingfrom a disease or disorder in which IL-13 activity is detrimental. Theinvention provides methods for reducing IL-13 activity in a subjectsuffering from such a disease or disorder, which method comprisesadministering to the subject an antibody or antibody portion of theinvention such that IL-13 activity in the subject is reduced.Preferably, the IL-13 is human IL-13, and the subject is a humansubject. Alternatively, the subject can be a mammal expressing an IL-13to which an antibody of the invention is capable of binding. Stillfurther the subject can be a mammal into which IL-13 has been introduced(e.g., by administration of IL-13 or by expression of an IL-13transgene). An antibody of the invention can be administered to a humansubject for therapeutic purposes. Moreover, an antibody of the inventioncan be administered to a non-human mammal expressing an IL-13 with whichthe antibody is capable of binding for veterinary purposes or as ananimal model of human disease. Regarding the latter, such animal modelsmay be useful for evaluating the therapeutic efficacy of antibodies ofthe invention (e.g., testing of dosages and time courses ofadministration).

As used herein, the term “a disorder in which IL-13 activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of IL-13 in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which IL-13activity is detrimental is a disorder in which reduction of IL-13activity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of IL-13 in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofIL-13 in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-IL-13 antibody as describedabove. Non-limiting examples of disorders that can be treated with theantibodies of the invention include those disorders discussed in thesection below pertaining to pharmaceutical compositions of theantibodies of the invention.

IL-13 has been implicated as having a pivotal role in causingpathological responses associated with asthma. However other mediatorsof differential immunological pathways are also involved in asthmapathogenesis, and blocking these mediators, in addition to IL-13, mayoffer additional therapeutic benefit. Thus, binding proteins of theinvention may be incorporated into DVD-Ig proteins where in the DVD iscapable of binding target pairs including, but not limited to, IL-13 anda pro-inflammatory cytokine, such as tumor necrosis factor-α (TNF-α).TNF-α may amplify the inflammatory response in asthma and may be linkedto disease severity (McDonnell, et al., Progress in Respiratory Research(2001), 31(New Drugs for Asthma, Allergy and COPD), 247-250.). Thissuggests that blocking both IL-13 and TNF-α may have beneficial effects,particularly in severe airway disease. In a preferred embodiment theDVD-Ig of the invention binds the targets IL-13 and TNFα and is used fortreating asthma.

In another embodiment binding proteins of the invention can be used togenerate DVD-Ig molecules that bind IL-13 and IL-1beta, IL-13 and IL-9;IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and ADAM8. Thepresent invention also provides DVD-Igs capable of binding IL-13 and oneor more targets involved in asthma selected from the group consisting ofCSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG,histamine and histamine receptors, IL1A, IL1B, 1L2, IL3, IL4, IL5, IL6,IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL14, IL15, IL16, IL17, IL18,IL19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28,IL-30, IL-31, IL-32, IL-33, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA,IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2,CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22,CCL24,CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21,TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, andChitinase.

D. Pharmaceutical Composition

The invention also provides pharmaceutical compositions comprising anantibody, or antigen-binding portion thereof, of the invention and apharmaceutically acceptable carrier. The pharmaceutical compositionscomprising antibodies of the invention are for use in, but not limitedto, diagnosing, detecting, or monitoring a disorder, in preventing,treating, managing, or ameliorating of a disorder or one or moresymptoms thereof, and/or in research. In a specific embodiment, acomposition comprises one or more antibodies of the invention. Inanother embodiment, the pharmaceutical composition comprises one or moreantibodies of the invention and one or more prophylactic or therapeuticagents other than antibodies of the invention for treating a disorder inwhich IL-13 activity is detrimental. Preferably, the prophylactic ortherapeutic agents known to be useful for or having been or currentlybeing used in the prevention, treatment, management, or amelioration ofa disorder or one or more symptoms thereof. In accordance with theseembodiments, the composition may further comprise of a carrier, diluentor excipient.

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidurala administration, intratumoral administration,and mucosal administration (e.g., intranasal and oral routes). Inaddition, pulmonary administration can be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. See,e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos.WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,each of which is incorporated herein by reference their entireties. Inone embodiment, an antibody of the invention, combination therapy, or acomposition of the invention is administered using Alkermes AIR®pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than anantibody of the invention of a subject to prevent, treat, manage, and/orameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent of theinvention can be delivered in a controlled release or sustained releasesystem. In one embodiment, a pump may be used to achieve controlled orsustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref.Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used to achieve controlled or sustained release of thetherapies of the invention (see e.g., Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983,J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howardet al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos. 5,679,377;5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO99/15154; and PCT Publication No. WO 99/20253. Examples of polymers usedin sustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In apreferred embodiment, the polymer used in a sustained releaseformulation is inert, free of leachable impurities, stable on storage,sterile, and biodegradable. In yet another embodiment, a controlled orsustained release system can be placed in proximity of the prophylacticor therapeutic target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity preferablygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entireties. In a specific embodiment, an antibody of theinvention, combination therapy, and/or composition of the invention isadministered using Alkermes AIR® pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompasses administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C. and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions of the invention is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml or at least 100 mg/ml. The liquid form should be storedat between 2° C. and 8° C. in its original container.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. Preferably, the antibody or antibody-portions will beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 2-4%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-Methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants. The pharmaceutical composition comprising theantibodies and antibody-portions of the invention prepared as aninjectable solution for parenteral administration, can further comprisean agent useful as an adjuvant, such as those used to increase theabsorption, or dispersion of a therapeutic protein (e.g., antibody). Aparticularly useful adjuvant is hyaluronidase, such as Hylenex®(recombinant human hyaluronidase). Addition of hyaluronidase in theinjectable solution improves human bioavailability following parenteraladministration, particularly subcutaneous administration. It also allowsfor greater injection site volumes (i.e. greater than 1 ml) with lesspain and discomfort, and minimum incidence of injection site reactions.(see WO2004078140, US2006104968 incorporated herein by reference).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingdisorders in which IL-13 activity is detrimental. For example, ananti-hIL-13 antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind othercytokines or that bind cell surface molecules). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

In certain embodiments, an antibody to IL-13 or fragment thereof islinked to a half-life extending vehicle known in the art. Such vehiclesinclude, but are not limited to, the Fc domain, polyethylene glycol, anddextran. Such vehicles are described, e.g., in U.S. application Ser. No.09/428,082 and published PCT Application No. WO 99/25044, which arehereby incorporated by reference for any purpose.

In a specific embodiment, nucleic acid sequences comprising nucleotidesequences encoding an antibody of the invention or another prophylacticor therapeutic agent of the invention are administered to treat,prevent, manage, or ameliorate a disorder or one or more symptomsthereof by way of gene therapy. Gene therapy refers to therapy performedby the administration to a subject of an expressed or expressiblenucleic acid. In this embodiment of the invention, the nucleic acidsproduce their encoded antibody or prophylactic or therapeutic agent ofthe invention that mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932(1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217;May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY (1993); and Kriegler, Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY (1990). Detailed description of variousmethods of gene therapy are disclosed in US20050042664 A1 which isincorporated herein by reference.

In another aspect, this application features a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing anIL-13-associated disorder, in a subject. The method includes:administering to the subject an IL-13 binding agent (particularly anantagonist), e.g., an anti-IL-13 antibody or fragment thereof asdescribed herein, in an amount sufficient to treat or prevent theIL-13-associated disorder. The IL-13 antagonist, e.g., the anti-IL-13antibody or fragment thereof, can be administered to the subject, aloneor in combination with other therapeutic modalities as described herein.

In one embodiment, the subject is a mammal, e.g., a human suffering fromone or more IL-13-associated disorders, including, e.g., respiratorydisorders (e.g., asthma (e.g., allergic and nonallergic asthma), chronicobstructive pulmonary disease (COPD), and other conditions involvingairway inflammation, eosinophilia, fibrosis and excess mucus production;atopic disorders (e.g., atopic dermatitis and allergic rhinitis);inflammatory and/or autoimmune conditions of, the skin, gastrointestinalorgans (e.g., inflammatory bowel diseases (IBD), such as ulcerativecolitis and/or Crohn's disease), and liver (e.g., cirrhosis, fibrosis);scleroderma; tumors or cancers, e.g., Hodgkin's lymphoma as describedherein. Accordingly, the disclosure includes the use of an IL-13 bindingagent (such as an anti-IL-13 antibody or fragment thereof describedherein) for a treatment described herein and the use of an IL-13 bindingagent (such as an anti-IL-13 antibody or fragment thereof describedherein) for preparing a medicament for a treatment described herein.

Examples of IL-13-associated disorders include, but are not limited to,a disorder chosen from one or more of: respiratory disorders, e.g.,asthma (e.g., allergic and nonallergic asthma (e.g., asthma due toinfection with, e.g., respiratory syncytial virus (RSV), e.g., inyounger children)), chronic obstructive pulmonary disease (COPD), andother conditions involving airway inflammation, eosinophilia, fibrosisand excess mucus production, e.g., cystic fibrosis and pulmonaryfibrosis; atopic disorders, e.g., resulting from an increasedsensitivity to IL-13 (e.g., atopic dermatitis, urticaria, eczema,allergic rhinitis, and allergic enterogastritis); inflammatoyv and/orautoimmune conditions of, the skin (e.g., atopic dermatitis),gastrointestinal organs (e.g., inflammatory bowel diseases (IBD), suchas ulcerative colitis and/or Crohn's disease), liver (e.g., cirrhosis,hepatocellular carcinoma), and scleroderma; tumors or cancers (e.g.,soft tissue or solid tumors), such as leukemia, glioblastoma, andlymphoma, e.g., Hodgkin's lymphoma; viral infections (e.g., fromHTLV-1); fibrosis of other organs, e.g., fibrosis of the liver, (e.g.,fibrosis caused by a hepatitis B and/or C virus); and suppression ofexpression of protective type 1 immune responses, (e.g., duringvaccination), as described herein.

In other embodiments, this application provides a method of treating(e.g., reducing, ameliorating) or preventing one or more symptomsassociated with a respiratory disorder, e.g., asthma (e.g., allergic andnonallergic asthma); allergies; chronic obstructive pulmonary disease(COPD); a condition involving airway inflammation, eosinophilia,fibrosis and excess mucus production, e.g., cystic fibrosis andpulmonary fibrosis. For example, symptoms of asthma include, but are notlimited to, wheezing, shortness of breath, bronchoconstriction, airwayhyperreactivity, decreased lung capacity, fibrosis, airway inflammation,and mucus production. The method comprises administering to the subjectan IL-13 antagonist, e.g., an IL-13 antibody or a fragment thereof, inan amount sufficient to treat (e.g., reduce, ameliorate) or prevent oneor more symptoms. The IL-13 antibody can be administered therapeuticallyor prophylactically, or both. The IL-13 antagonist, e.g., the anti-IL-13antibody, or fragment thereof, can be administered to the subject, aloneor in combination with other therapeutic modalities as described herein.Preferably, the subject is a mammal, e.g., a human suffering from anIL-13-associated disorder as described herein.

In another aspect, this application provides a method for detecting thepresence of IL-13 in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, biopsy). The subject method can be used todiagnose a disorder, e.g., an immune cell-associated disorder. Themethod includes: (i) contacting the sample or a control sample with theanti-IL-13 antibody or fragment thereof as described herein; and (ii)detecting formation of a complex between the anti-IL-13 antibody orfragment thereof, and the sample or the control sample, wherein astatistically significant change in the formation of the complex in thesample relative to the control sample is indicative of the presence ofthe IL-13 in the sample.

In yet another aspect, this application provides a method for detectingthe presence of IL-13 in vivo (e.g., in viva imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., anIL-13-associated disorder. The method includes: (i) administering theanti-IL-13 antibody or fragment thereof as described herein to a subjector a control subject under conditions that allow binding of the antibodyor fragment to IL-13; and (ii) detecting formation of a complex betweenthe antibody or fragment and IL-13, wherein a statistically significantchange in the formation of the complex in the subject relative to thecontrol subject is indicative of the presence of IL-13.

Antibodies of the invention, or antigen binding portions thereof can beused alone or in combination to treat such diseases. It should beunderstood that the antibodies of the invention or antigen bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agent thatimparts a beneficial attribute to the therapeutic composition e.g., anagent which effects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The combination therapy can include one or more IL-13 antagonists, e.g.,anti-IL-13 antibodies or fragments thereof, coformulated with, and/orcoadministered with, one or more additional therapeutic agents, e.g.,one or more cytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents (e.g., systemic anti-inflammatory agents),anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors, and/orcytotoxic or cytostatic agents, as described in more herein.

Examples of preferred additional therapeutic agents that can becoadministered and/or coformulated with one or more IL-13 antagonists,e.g., anti-IL-13 antibodies or fragments thereof, include, but are notlimited to, one or more of: inhaled steroids; beta-agonists, e.g.,short-acting or long-acting beta-agonists; antagonists of leukotrienesor leukotriene receptors; combination drugs such as ADVAIR; IgEinhibitors, e.g., anti-IgE antibodies (e.g., XOLAIR); phosphodiesteraseinhibitors (e.g., PDE4 inhibitors); xanthines; anticholinergic drugs;mast cell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or itsreceptors including H1, H2, H3, and H4, and antagonists of prostaglandinD or its receptors (DP1 and CRTH2). Such combinations can be used totreat asthma and other respiratory disorders. Additional examples oftherapeutic agents that can be coadministered and/or coformulated withone or more anti-IL-13 antibodies or fragments thereof include one ormore of: TNF antagonists (e.g., a soluble fragment of a TNF receptor,e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kDTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL)); TNF enzymeantagonists, e.g., TNF converting enzyme (TACE) inhibitors; muscarinicreceptor antagonists; TGF-beta antagonists; interferon gamma;perfenidone; chemotherapeutic agents, e.g., methotrexate, leflunomide,or a sirolimus (rapamycin) or an analog thereof, e.g., CCI-779; COX2 andcPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors, TPL-2, MK-2and NFkB inhibitors, among others.

Other preferred combinations are cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-31, interferons, EMAP-II,GM-CSF, FGF, EGF, PDGF, and edothelin-1, as well as the receptors ofthese cytokines and growth factors. Antibodies of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligandsincluding CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the inflammatory cascade; preferred examples include TNFantagonists like chimeric, humanized or human TNF antibodies, D2E7, (PCTPublication No. WO 97/29131), CA2 (Remicade™), CDP 571, and soluble p55or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) orp55TNFR1gG (Lenercept), and also TNF converting enzyme (TACE)inhibitors; similarly IL-1 inhibitors (Interleukin-1-converting enzymeinhibitors, IL-1RA etc.) may be effective for the same reason. Otherpreferred combinations include Interleukin 4. Yet another preferredcombination are other key players of the asthmatic response which mayact parallel to, dependent on or in concert with IL-13 function;especially preferred are IL-9 antagonists including IL-9 antibodies. Ithas been shown that IL-13 and IL-9 have overlapping but distinctfunctions and a combination of antagonists to both may be mosteffective. Yet another preferred combination are anti-IL-5 antibodies.Yet other preferred combinations include antagonists of chemokinesincluding MCP-1, MCP-4, eotaxins, RANTES, MDC, CCL-12 and CCL-17 (TARC)and chemokine receptors including CCR2, CCR3, CCR4, and CXCR4. Yetcombinations can include antagonists to asthma mediators including acidmammalian chitinase, CRHT2, chymase, S1P1, S1P2, Tyk2, ROCKII, Stat6,p38, NFkB, phosphodiesterase 4 (PDE-4), mast cell trytase, NO,adenosine, IKK2, GATA3, ICAM-1, VCAM-1, and ICOS.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to benoted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1 Generation and Isolation of Anti Human IL-13Monoclonal Antibodies Example 1.1 Assays to Identify Anti Human IL-13Antibodies

Throughout Example 1 the following assays were used to identify andcharacterize anti human IL-13 antibodies unless otherwise stated.

Example 1.1.A ELISA

Enzyme Linked Immunosorbent Assays to screen for antibodies that bindhuman IL-13 were performed as follows.

ELISA plates (Corning Costar, Acton, Mass.) were coated with 50 μL/wellof 5 μg/ml goat anti-mouse IgG Fc specific (Pierce # 31170, Rockford,Ill.) in Phosphate Buffered Saline (PBS) overnight at 4 degrees Celsius.Plates were washed once with PBS containing 0.05% Tween-20. Plates wereblocked by addition of 200 μL/well blocking solution diluted to 2% inPBS (BioRad #170-6404, Hercules, Calif.) for 1 hour at room temperature.Plates were washed once after blocking with PBS containing 0.05%Tween-20.

Fifty microliters per well of mouse sera or hybridoma supernatantsdiluted in PBS containing 0.1% Bovine Serum Albumin (BSA) (Sigma, St.Louis, Mo.) was added to the ELISA plate prepared as described above andincubated for 1 hour at room temperature. Wells were washed three timeswith PBS containing 0.05% Tween-20. Fifty microliters of biotinylatedrecombinant purified human IL-13 variant (R110Q) diluted to 100 ng/mL inPBS containing 0.1% BSA was added to each well and incubated for 1 hourat room temperature. Plates were washed 3 times with PBS containing0.05% Tween-20. Streptavidin HRP (Pierce #21126, Rockland, Ill.) wasdiluted 1:20000 in PBS containing 0.1% BSA; 50 μL/well was added and theplates incubated for 1 hour at room temperature. Plates were washed 3times with PBS containing 0.05% Tween-20. Fifty microliters of TMBsolution (Sigma # T0440, St. Louis, Mo.) was added to each well andincubated for 10 minutes at room temperature. The reaction was stoppedby addition of 1N sulphuric acid. Plates were read spectrophotmetricallyat a wavelength of 450 nm.

Example 1.1.B Affinity Determinations Using BIACORE Technology

The BIACORE assay (Biacore, Inc, Piscataway, N.J.) determines theaffinity of antibodies with kinetic measurements of on-, off-rateconstants. Binding of antibodies to recombinant purified human IL-13 orrecombinant purified human IL-13 variant (R110Q) were determined bysurface plasmon resonance-based measurements with a Biacore® 3000instrument (Biacore® AB, Uppsala, Sweden) using running HBS-EP (10 mMHEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at25° C. All chemicals were obtained from Biacore® AB (Uppsala, Sweden) orotherwise from a different source as described in the text.Approximately 5000 RU of goat anti-mouse IgG, (Fcγ), fragment specificpolyclonal antibody (Pierce Biotechnology Inc, Rockford, Ill.) dilutedin 10 mM sodium acetate (pH 4.5) was directly immobilized across a CM5research grade biosensor chip using a standard amine coupling kitaccording to manufacturer's instructions and procedures at 25 μg/ml.Unreacted moieties on the biosensor surface were blocked withethanolamine. Modified carboxymethyl dextran surface in flowcell 2 and 4was used as a reaction surface. Unmodified carboxymethyl dextran withoutgoat anti-mouse IgG in flow cell 1 and 3 was used as the referencesurface. For kinetic analysis, rate equations derived from the 1:1Langmuir binding model were fitted simultaneously to association anddissociation phases of all eight injections (using global fit analysis)with the use of Biaevaluation 4.0.1 software. Purified antibodies werediluted in HEPES-buffered saline for capture across goat anti-mouse IgGspecific reaction surfaces. Mouse antibodies to be captured as a ligand(25 μg/ml) were injected over reaction matrices at a flow rate of 5μl/min. The association and dissociation rate constants, k_(on) (unitM⁻¹s⁻¹) and k_(off) (unit s⁻¹) were determined under a continuous flowrate of 25 μl/min Rate constants were derived by making kinetic bindingmeasurements at ten different antigen concentrations ranging from 10-200nM. The equilibrium dissociation constant (unit M) of the reactionbetween mouse antibodies and recombinant purified human IL-13 orrecombinant purified human IL-13 was then calculated from the kineticrate constants by the following formula: K_(D)=k_(off)/k_(on). Bindingis recorded as a function of time and kinetic rate constants arecalculated. In this assay, on-rates as fast as 10⁶M⁻¹s⁻¹ and off-ratesas slow as 10⁻⁶ s⁻¹ can be measured.

Example 1.1.C Functional Activity of Anti Human IL-13 Antibodies

To examine the functional activity of the anti-human IL-13 antibodies ofthe invention, the antibodies were used in the following assays thatmeasure the ability of an antibody to inhibit IL-13 activity.

Example 1.1.C1 A-549 Bioassay

The ability of anti-human IL-13 antibodies to inhibit the human IL-13induced production of TARC(CCL-17) by A-549 cells was analyzed asfollows. A-549 cells were seeded on day one in 96-well plate (2E5cells/well) in RPMI growth medium (with 10% FBS). On day two, the mediumwas replaced with fresh RPMI growth medium containing 400 ng/ml rhTNF(100 μl/well). Meanwhile, various concentrations of immunized mouseserum, murine hybridoma supernatant or purified anti-human IL-13antibodies were preincubated for one hour at 37° C. with 10 ng/mlrecombinant purified human IL-13 or IL-13 variant in 100 μL RPMIcomplete medium in a microtiter plate (U-bottom, 96-well, Costar). Theantibody plus recombinant purified human IL-13 mixture was then added(100 μl/well) to the TNF-treated A-549 cells, with the final volume of200 μl/well (final IL-13 and TNF concentrations were 5 ng/ml and 200ng/ml, respectively), and incubated for 18 hours at 37° C. Afterincubation, 150 μL of cell-free supernatant was withdrawn from each welland the level of human TARC produced was measured using a human TARCELISA (R&D Systems Cat#DDN00).

The A-549 cells also respond to IL-13 of other species, includingcynomolgus monkey, mouse, rat, and sheep, with ED₅₀ values similar tothat of human IL-13. Therefore it was employed for cross-reactiveanalysis of anti-hIL-13 mAbs to IL-13 of other species using the sameexperimental protocol.

Example 1.2 Generation of Anti Human IL-13 Monoclonal Antibodies

Anti human IL-13 mouse monoclonal antibodies were obtained as follows:

Example 1.2.A Immunization of Mice with Human IL-13 Antigen

Twenty micrograms of recombinant purified human IL-13 variant(Peprotech) mixed with complete Freund's adjuvant or Immunoeasy adjuvant(Qiagen, Valencia, Calif.) was injected subcutaneously into five 6-8week-old Balb/C, five C57B/6 mice, and five AJ mice on Day 1. On days24, 38, and 49, twenty micrograms of recombinant purified human IL-13variant mixed with incomplete Freund's adjuvant or Immunoeasy adjuvantwas injected subcutaneously into the same mice. On day 84 or day 112 orday 144, mice were injected intravenously with 1 ug recombinant purifiedhuman IL-13 variant.

Example 1.2.B Generation of Hebridoma

Splenocytes obtained from the immunized mice described in Example 1.2.Awere fused with SP2/O—Ag-14 cells at a ratio of 5:1 according to theestablished method described in Kohler, G. and Milstein 1975, Nature,256:495 to generate hybridomas. Fusion products were plated in selectionmedia containing azaserine and hypoxanthine in 96-well plates at adensity of 2.5×10⁶ spleen cells per well. Seven to ten days post fusion,macroscopic hybridoma colonies were observed. Supernatant from each wellcontaining hybridoma colonies was tested by ELISA for the presence ofantibody to IL-13 variant (as described in Example 1.1.A). Supernatantsdisplaying IL-13 variant-specific activity were then tested for theability to neutralize IL-13 variant and IL-13 wild-type in the A-549bioassay for TARC (as described in Example 1.1.C).

Example 1.2.C Identification and Characterization of Anti Human IL-13Monoclonal Antibodies

Hybridomas producing antibodies that bound IL-13 variant, generatedaccording to Examples 1.2.B and 1.2.C, and capable of binding IL-13variant specifically and particularly those with IC₅₀ values in theA-549 bioassay of 5 nM or less than 5 nM were scaled up and cloned bylimiting dilution.

Hybridoma cells were expanded into media containing 10% low IgG fetalbovine serum (Hyclone #SH30151, Logan, Utah). On average, 250 mL of eachhybridoma supernatant (derived from a clonal population) was harvested,concentrated and purified by protein A affinity chromatography, asdescribed in Harlow, E. and Lane, D. 1988 “Antibodies: A LaboratoryManual”. The ability of purified mAbs to inhibit IL-13 activity wasdetermined using the A-549 bioassay as described in Examples 1.1.C.Table 7 shows IC₅₀ values from the A-549 bioassays for 17 monoclonalantibodies.

TABLE 7 Neutralization of IL-13 by anti IL-13 mAbs in A-549 bioassayMurine Average IC₅₀ (nM) Average IC₅₀ (nM) Monoclonal Human IL-13 HumanIL-13 Average IC₅₀ (nM) Antibody Isotype wild-type variant CynomolgusIL-13 4A8 IgG1λ ND 2.70E−10 ND 6C8 IgG1κ 7.20E−10 3.40E−10 1.61E−10 5F1IgG1κ 9.70E−11 9.00E−11 1.88E−09 1B6 IgG1κ 8.40E−10 2.40E−10 5.21E−105G1 IgG1κ 7.60E−11 4.80E−11 6.12E−10 29G5 IgG2aκ 2.90E−10 2.00E−104.39E−09 33C3 IgG1κ 1.50E−10 1.00E−10 8.47E−10 25C8 IgG1κ 2.30E−102.60E−10 1.88E−10 13C5 IgG1κ 1.90E−10 1.70E−10 5.00E−09 3E5 IgG2aκ1.30E−10 3.00E−10 1.61E−10 3H7 IgG2aκ NA 5.80E−10 7.97E−10 5D3 IgG1κ7.05E−10 2.90E−10 2.91E−10 8B6 IgG1κ ND 4.80E−10 3.95E−10 21D9 IgG2bκ6.82E−11 1.36E−10 3.40E−10 14B2 IgG1κ ND 4.36E−10 NA 9C11 IgG1κ 1.06E−101.70E−10 6.40E−10 22D10 IgG1κ 2.84E−10 5.40E−10 6.11E−09

The binding affinities of the monoclonal antibodies to recombinantpurified human IL-13 variant and wild-type were determined using surfaceplasmon resonance (Biacore®) measurement as described in Example 1.1.B.Table 8 shows the affinity of the 18 monoclonal antibodies describedabove for human IL-13.

TABLE 8 Affinity of anti IL-13 mAbs for human wild-type and variantIL-13 Human wild-type IL-13 Human variant IL-13 mAb k_(on) (1/M · s)k_(off) (1/s) K_(D) (M) k_(on) (1/M · s) k_(off) (1/s) K_(D) (M) 4A88.90E−11 1.57E−10 6C8 1.45E+06 7.02E−04 4.84E−10 9.78E+05 3.94E−044.03E−10 5F1 7.74E+05 2.24E−05 2.89E−11 5.02E+05 1.57E−05 3.14E−11 1B69.51E+05 5.18E−04 5.45E−10 1.06E+05 2.22E−04 2.10E−10 5G1 6.26E+055.49E−06 8.77E−12 1.57E+05 2.05E−05 1.30E−10 29G5 8.59E+05 1.75E−042.04E−10 3.16E+05 1.04E−04 3.29E−10 33C3 2.33E+06 1.49E−04 6.39E−117.70E+05 9.59E−05 1.24E−10 25C8 3.45E+05 2.60E−05 7.54E−11 1.34E+058.45E−06 6.31E−11 13C5 1.25E+06 9.31E−05 7.45E−11 5.74E+05 4.35E−057.59E−11 3E5 1.44E+06 6.58E−04 4.57E−10 1.85E+06 4.68E−04 2.53E−10 3H7NB 2.54E+05 5.58E−05 2.20E−10 5D3 1.63E+06 4.83E−04 2.96E−10 1.51E+065.84E−04 3.87E−10 8B6 1.16E+06 6.07E−04 5.23E−10 9.83E+05 9.60E−049.76E−10 21D9 8.52E+05 6.58E−05 7.72E−11 8.31E+05 6.18E−05 7.44E−11 14B26.69E+05 1.84E−04 2.75E−10 8.08E+05 2.79E−04 3.46E−10 9C11 5.79E+056.50E−05 1.12E−10 6.37E+05 5.86E−05 9.21E−11 22D10 1.82E+05 6.50E−053.56E−10 2.45E+05 1.55E−04 6.32E−10

Example 1.2.C.1 Species Specificity of Murine Monoclonal Anti-HumanIL-13 Antibodies

To determine whether the 17 monoclonal antibodies described aboverecognize murine IL-13, an indirect ELISA was set up by coating ELISAplates with 5 ug/ml of goat anti-mouse IgG, Fc fragment specificantibody (Pierce #31170, Rockland, Ill.). Murine anti-human IL-13 mAbswere prepared at various concentrations ranging from 0.1 to 100 ng/ml inPBS containing 0.1% BSA; 50 ul of each antibody dilution was added tothe coated ELISA plate and incubated for 1 hour at room temperature.Wells were washed 3 times with PBS containing 0.05% Tween-20.Recombinant biotinylated mouse IL-13 (R&D Systems) was diluted at 0.1ug/ml in PBS containing 0.1% BSA; 50 ul/well was added and the platesincubated for 1 hour at room temperature. Wells were washed 3 times withPBS containing 0.05% Tween-20. Streptavidin HRP (Pierce #21126,Rockland, Ill.) was diluted 1:20000 in PBS containing 0.1% BSA; 50mL/well was added and the plates incubated for 1 hour at roomtemperature. Plates were washed 3 times with PBS containing 0.05%Tween-20. Fifty microliters of TMB solution (Sigma # T0440, St. Louis,Mo.) was added to each well and incubated for 10 minutes at roomtemperature. The reaction was stopped by addition of 1N sulphuric acid.Plates were read spectrophotmetrically at a wavelength of 450 nm.Results from the indirect ELISA indicated that mAb 3H7 was able to bindmIL-13. In subsequent bioassay it was shown that 3H7 could inhibitmIL-13-stimulated TARC production in a dose-dependent mannor, with anIC₅₀ of 2.4 nM. Biacore analysis also demonstrated positive binding of3H7 to mIL-13, with a K_(D) of 12 nM. All other mAbs in table 8 did notshow any positive binding to mouse IL-13.

Neutralisation potency of anti-hIL-13 mAbs against non-human primate(cynomolgus) IL-13 and sheep IL-13 were also measured in the A-548bioassay. To generate cyno and sheep IL-13, cDNA for each protein wasobtained by PCR on genomic DNA template using degenerate primers basedon the human IL-13 sequence. Recombinant cyno and sheep IL-13 proteinswere subsequently expressed in transiently transfected COS cells.Wild-type human IL-13 was also generated in parallel as a control in allfunctional studies. A-549 cells responded to both cyno and sheep IL-13with a similar ED₅₀ to that of human IL-13. Most of the mAbs neutralizedactivity of cyno IL-13, demonstrating cross-reactivity to cyno IL-13(Table 7). However none of the antibodies showed significantneutralization of sheep IL-13.

Example 1.2.C.2 Murine Monoclonal Anti-Human IL-13 Antibodies BlockIL-13 Binding to IL-13 Receptors (IL-13Rα1 and IL-13Rα2)

IL-13 activity is mediated through a receptor complex consisting of theIL-13Rα1 and IL-4Rα chains. The cytokine first undergoes a relativelylow affinity interaction with IL-13Rα1 on the surface of cells. TheIL-13/IL-13Rα1 complex then recruits IL-4Rα to form the complete IL-13receptor, which is bound to its ligand (IL-13) with high affinity(Zurawski et al. (1993) EMBO J. 12:2663; Zurawski et al. (1995) J. Biol.Chem. 270:23869). The binding of IL-13 with the high affinity receptorthen sends downstream signals through the IL-4Rα chain involving theJanus kinase-signal transducer and activator of transcription (JAK-STAT)pathway, e.g., via phosphorylation of STAT6, which can be monitored asone of the earliest cellular responses to IL-13 (Murata et al., supra).

There is another IL-13-binding receptor, the IL-13Rα2 chain (IL-13Rα2),which binds to IL-13 with high affinity (0.25-1.2 nM) (Caput, et al 1996J. Biol. Chem. 271, 16921-16926; Donaldson et al 1998 J. Immunol. 161,2317-2324). No other receptor molecule is known to be involved in theIL-13/IL-13Rα2 complex. IL-13Rα2 was initially thought to act as anonsignaling “decoy” receptor. However, it was later discovered that itcan bind to IL-13, and signals through AP-1 pathway, leading to TGF-betaproduction in certain cell types including macrophages, which in turnleads to lung fibrosis (Fichtner-Feigl. 2006 Nat Med 12:99-106).Therefore, both IL-13Rα1/IL-4R complex and IL-13Rα2 pathways contributeto the overall pathophysiology of asthma and other IL-13 mediateddiseases. Several approaches, such as epitope mapping, receptor bindingassays, size exclusion chromatography (SEC), and further BIACOREanalysis, were used to elucidate the interaction between the anti-IL-13antibodies of the invention and human IL-13.

To determine whether the monoclonal antibodies described above are ableto block IL-13 binding to IL-13 receptors (IL-13Rα1 and IL-13Rα2), areceptor binding ELISA was developed as follows. High-binding 96-wellELISA plates were coated with 4 ug/ml of recombinant IL-13Rα1/Fc orIL-13Rα2/Fc (R&D Systems) in 100 ul/well coating buffer(Carbonate-bicarbonate buffer, Pierce) at 4° C. After 16 hr, coatingsolution was removed by flicking plate contents in sink, and plates werewashed and blocked 4 times with Superblock Blocking Buffer (240 ul/well)(Pierce). Anti-IL13 mAbs (1:4 serially diluted from 40 ug/ml, 50ul/well) and Biotin-IL-13 (50 ul/well, final concentrations of 5 nM forhIL-13Rα1/Fc, and 0.5 nM for hIL-13Rα2/Fc) were added and incubated for2 hr at room temperature (RT). Plates were washed 5 times with 300 ul0.1% PBST, and then 100 ul of 1:5000 diluted mouse anti-Biotin MAb(Jackson Immunosciences) was added and incubated at RT for 45 min. Theplates were washed again 5 times with 300 ul 0.1% PBST, followed byaddition of TMB substrate reagent (100 ul/well, Pharmingen); developedfor 5 min, and stopped by adding 50 ul of 2M H2SO4 (VWR). ODs at 450 nmwere determined by spectrophotometry.

Additionally, the receptor blocking properties of the mAbs were alsoassessed by receptor binding assay using IL-13Rα2-transfected COS cells.Recombinant human IL-13 was labeled with ¹²⁵I (Amersham, ArlingtonHeights, Ill.), using IODO-GEN reagent (Pierce, Rockford, Ill.) aspreviously described (Obiri N I et al., (1995) J Biol Chem.270:8797-8804). The specific activity of the radiolabeled IL-13 wasestimated to be 158 μCi/μg protein. The labeled IL-13 exhibited similarbioactivity as unlabeled IL-13, as assessed by the A-549 bioassay. Forbinding experiments, COS cells were transiently transfected with humanIL-13Rα2 by Lipofectamine 2000 (Invitrogen), and incubated for 48 hr.Transfected COS cells (5×105 cells in 100 μL binding buffer: RPMI 1640containing 0.2% human serum albumin and 10 mmol HEPES) were incubatedwith 1.0 nM ¹²⁵I-IL-13 with or without 1 uM unlabeled IL-13 at 4° C. for2 hours. Cell-bound ¹²⁵I-IL-13 was separated from unbound ¹²⁵I-IL-13 bycentrifugation through a phthalate oil gradient, and radioactivity wasdetermined with a gamma counter (Wallac, Gaithersburg, Md.). Forantibody displacement assay, transfected COS cells were incubated with125I-IL-13 (1.0 nM) with or without increasing concentrations (up to 50ug/ml) of anti-IL-13 antibodies, as described above. Both forms ofreceptor binding assay demonstrated the following: First, 13C5 and 9C11blocked IL-13 binding to IL-13Rα1; second, 13C5 strongly blocked IL-13binding to IL-13Rα2 (IC₅₀˜1-3 nM in both cell surface RBA and RB ELISA),whereas 9C11 blocked IL-13 binding of IL-13Rα2 with a lower potency(IC₅₀>10 nM); and third, 5G1 and 3E5 failed to block IL-13 binding toeither IL-13Rα1 or IL-13Rα2. Three other anti-IL-13 antibodies, BAK502G9(CAT PCT WO 2005/007699), mAb13.2 (Wyeth PCT WO 2005/123126A2) and MJ2-7(Wyeth PCT WO 2006/0073148A1) were also analyzed for their ability toblock human IL-13 binding to human IL-13 Rα2 on both receptor bindingELISA and cell surface RBA. Antibody mAb13.2 did not block IL-13 bindingto eith IL-13Rα1 or IL-13Rα2. BAK502G9 and MJ2-7 was able to block IL-13binding to IL-13Rα1; however they exhibited low potency in blockingIL-13 binding to IL-13Rα2 with antibody concentrations up to 50 μg/ml(330 nM).

The interaction between IL-13 and IL-13Rα1/α2 in the presense ofanti-IL-13 mAbs was also analyzed by BIACORE. This analysis was done inseveral formats. First, IL-13Rα1/Fc was bound to the Biacore chip andIL-13 was flowed over the chip, in the presence and absence ofanti-IL-13 mAbs. MAbs 13C5 and 9C11, among others, were able to blockIL-13 binding to IL-13Rα1, whereas 5G1 and 3E5 failed to inhibit IL-13binding to IL-13Rα1, consistent with the receptor binding assays.Second, IL-4R was bound to the BIACORE chip, and a complex of IL-13prebound to IL-13Rα1 was flowed over the chip. In absence of anti-IL-13mAbs, formation of a trimolecular complex was demonstrated. However,addition of anti-IL-13 antibody 5G1 to the mixture of IL-13 prebound toIL-13Rα1 prevented binding to IL-4R on the chip. This indicated that,even though 5G1 could not block IL-13 binding to IL-13Rα1, it couldblock binding of IL-13 binding to IL-4R, providing a mechanistic basisfor its IL-13-neutralizing activity. These observations were furtherconfirmed by size exclusion chromatography (SEC), where hetero-trimericcomplexes (mAb-IL-13-IL-13Rα1/Fc) were observed for 5G1, but not for13C5. Subsequent epitope mapping studies using proteinase processing ofmAb-IL-13 complex followed by mass spec analysis indicated thefollowing: First, 5G1 binds to IL-13 residues including the N-terminal11-aa peptide (GPVPPSTALRE), covering part of the Helix-A region thathas been shown to interact with IL-4R (Moy et al 2001 J Mol Biol.310:219 and Horita et al., (2001) J Mol Biol. 310:231); second, antibody9C11 interacts with a region between Helix C and Helix D (VSAGQFSSLHVR);and third, antibody 13C5 interacts with IL-13 residues including aregion covering Helix D (VRDTK IEVAQ FVKDL LLHLK KLFRE GR correspondingto amino acid 104-130 of SEQ ID NO. 1). Helix D has been shown tointeract with IL-13 receptors (Moy et al 2001 J Mol Biol. 310:219;Horita et al 2001 J Mol Biol. 310:231; and Madhankumar et al 2002 JBC277:43194). Since 13C5 binds human IL-13 variant (K_(D)=50 pM) much morestrongly than cynomolgus IL-13 (K_(D)=1800 pM), and the only sequencedifference between human IL-13 variant and cynomolgus IL-13 within thispotential 13C5 epitope region is L in human but V in cyno IL-13 atposition 120, we generated a V120L mutant cyno IL-13 and tested whetherthis mutation will have increased binding affinity to 13C5 over wildtype cyno IL-13. Based on Biacore and bioassay results, the bindingaffinity as well as neutralization potency of 13C5 for the V120L mutantcyno IL-13 were equivalent to that for the wild type cyno IL-13,indicating that this V/L difference at position 120 within theC-terminal region does not contribute to the 13C5 affinity differencefor human IL-13 variant versus cyno IL-13, and that there must be otherresidues outside the C-terminal region that contribute to thedifferential binding affinity of 13C5 to human and cyno IL-13. This isconsistent with the observation that 13C5 does not recognize denaturedhuman IL-13 by Western blot analysis, indicating that the bindingepitope of 13C5 on human IL-13 is strongly conformational.

The binding and epitope mapping studies indicated that 5G1 did notinhibit IL-13 interaction with IL-13Rα1 but disrupted the interaction ofIL-13/IL-13Rα1 with IL-4Rα. This disruption is thought to interfere withthe formation of a functional IL-13 signaling complex. Theseobservations provide a theoretical model for the neutralizing activityof this antibody in an IL-13Rα1/IL-4R-mediated system such as A-549cells. In contrast, 13C5 blocked binding of IL-13 to both IL-13Rα1 andIL-13Rα2. Interestingly, even though 9C11 was able to block IL-13binding to IL-13Rα1, it only showed partial (or low potency) inhibitionof IL-13 binding to IL-13Rα2. Although IL-13Rα1 and Rα2 adopt similar3-dimentional fold and IL-13 binding orientation, they have a lowsequence identity and therefore specific residues responsible for IL-13binding may vary (Arima 2005 JBC 280:24915; and Madhankumar et al 2002JBC 277:43194). Consequently, specific residues on IL-13 for binding toIL-13Rα1 and IL-13Rα2 may differ, which could explain the differentialreceptor blocking properties of 9C11.

The receptor binding assays, epitope mapping, Biacore, and bioassaydescribed above collectively indicate that a neutralizing anti-IL-13antibody can inhibit IL-13-associated activity through the followingmechanisms:

1) Inhibit IL-13 binding to both IL-13Rα1 and IL-13Rα2 by interactingwith IL-13 in the region involved in receptor binding to both IL-13Rα1and IL-13Rα2. An example of such an antibody is 13C5. Such antibodieswill inhibit IL-13 signalling through both IL-13Rα1/IL-4R complex andIL-13Rα2.

2) Does not inhibit IL-13 binding to either IL-13Rα1 or IL-13Rα2.However the antibody inhibits interaction with IL-4 receptor, therefore,inhibits IL-13 signalling through IL-13Rα1/IL-4R complex. Such antibodymay not inhibit IL-13Rα2 signalling. Examples of such antibodies are 5G1and mAb13.2 (Wyeth PCT WO 2005/123126).

3) Inhibits IL-13 binding to IL-13Rα1 but not effectively inhibit IL-13binding to IL-13Rα2. This could occur for the following reasons: a)epitope: a region that involved in IL-13Rα1 binding but not in IL-13Rα2binding or not as strongly involved in IL-13Rα2 binding. An example is9C11; b) affinity: since IL-13Rα2 has much higher affinity than IL-13Rα1for IL-13, a low affinity antibody may be able to block IL-13 binding toIL-13Rα1 but not to IL-13Rα2 at physiological concentrations of atherapeutic antibody. An example is BAK502G9, which displayed a 2.11 nMaffinity to recombinant wild-type human IL-13 as assessed by Biacore(CAT PCT WO 2005/007699). Another example is MJ2-7, which displayed a1.4 nM affinity to recombinant wild-type human IL-13 and a higheraffinity (43 pM) for monkey IL-13 as assessed by Biacore (Wyeth PCT WO2006/0073148A1). Due to the affinity difference, this mAb can inhibitmonkey IL-13 binding to IL-13Rα2 effectively; however it inhibits humanIL-13 binding to the same receptor with much less potency.

The mAbs BAK502G9 and MJ2-7 have similar epitopes (CAT PCT WO2005/007699 and Wyeth PCT WO 2006/0073148A1) and they compete forbinding to IL-13 as assessed by competition ELISA. Briefly, BAK502G9 wasimmobilized on ELISA plate followed by wash and blocking. Thenbiotinylated human IL-13 (10 ng/ml) was added to the plate in thepresence of various concentrations of MJ2-7 (0.2 ng/ml to 20 ug/ml),followed by wash and detection using HRP-conjugated anti-biotinantibody. This study demonstrated that MJ2-7 dose-dependently competedwith BAK502G9 for binding to human IL-13. A negative control IgG did notshow competition with BAK502G9.

Example 1.2.D Determination of the Amino Acid Sequence of the VariableRegion for Each Murine Anti-human IL-13 mAb

For each amino acid sequence determination, approximately 10×10⁶hybridoma cells were isolated by centrifugation and processed to isolatetotal RNA with Trizol (Gibco BRL/Invitrogen, Carlsbad, Calif.) followingmanufacturer's instructions. Total RNA was subjected to first strand DNAsynthesis using the SuperScript First-Strand Synthesis System(Invitrogen, Carlsbad, Calif.) per the manufacturers instructions.Oligo(dT) was used to prime first-strand synthesis to select forpoly(A)⁺ RNA. The first-strand cDNA product was then amplified by PCRwith primers designed for amplification of murine immunoglobulinvariable regions (Ig-Primer Sets, Novagen, Madison, Wis.). PCR productswere resolved on an agarose gel, excised, purified, and then subclonedwith the TOPO Cloning kit into pCR2.1-TOPO vector (Invitrogen, Carlsbad,Calif.) and transformed into TOP10 chemically competent E. coli(Invitrogen, Carlsbad, Calif.). Colony PCR was performed on thetransformants to identify clones containing insert. Plasmid DNA wasisolated from clones containing insert using a QIAprep Miniprep kit(Qiagen, Valencia, Calif.). Inserts in the plasmids were sequenced onboth strands to determine the variable heavy or variable light chain DNAsequences using M13 forward and M13 reverse primers (Fermentas LifeSciences, Hanover Md.). Variable heavy and variable light chainsequences of the 17 monoclonal antibodies described in Example 1.2.0 aredescribed in Table 5.

Example 2 Recombinant Anti Human IL-13 Antibodies Example 2.1Construction and Expression of Recombinant Chimeric Anti Human IL-13Antibodies

The DNA encoding the heavy chain constant region of murine anti-humanIL-13 monoclonal antibodies 5G1, 13C5, 9C11, 21D9, and 3H7 was replacedby a cDNA fragment encoding the human IgG1 constant region containing 2hinge-region amino acid mutations by homologous recombination inbacteria. These mutations are a leucine to alanine change at position234 (EU numbering) and a leucine to alanine change at position 235 (Lundet al., 1991, J. Immunol., 147:2657). The light chain constant region ofeach of these antibodies was replaced by a human kappa constant region.Full-length chimeric antibodies were transiently expressed in COS cellsby co-transfection of chimeric heavy and light chain cDNAs ligated intothe pBOS expression plasmid (Mizushima and Nagata, Nucleic AcidsResearch 1990, Vol 18, pg 5322). Cell supernatants containingrecombinant chimeric antibody were purified by Protein A Sepharosechromatography and bound antibody was eluted by addition of acid buffer.Antibodies were neutralized and dialyzed into PBS.

The purified chimeric anti-human IL-13 monoclonal antibodies were thentested for their ability to inhibit the IL-13 induced production of TARCby A-549 cells as described in Examples 1.1.C2 and 1.1.C3. Table 12shows IC₅₀ values from the A-549 bioassays for three chimericantibodies.

TABLE 9 Neutralization of rhIL-13 wt by anti IL-13 Chimeric Antibodiesin A-549 bioassay Chimeric Average IC₅₀ (M) 5G1-Chim 4.10E−11 13C5-Chim1.91E−10 9C11-Chim 1.23E−10

Example 2.2 Construction and Expression of Humanized Anti Human IL-13Antibodies Example 2.2.1 Selection of Human Antibody Frameworks

Each murine variable heavy and variable light chain gene sequence (asdescribed in Table 3) was separately aligned against 44 humanimmunoglobulin germline variable heavy chain or 46 germline variablelight chain sequences (derived from NCBI Ig Blast website athttp://www.ncbi.nlm.nih.gov/igblast/retrieveig.html.) using Vector NTIsoftware.

Humanization was based on amino acid sequence homology, CDR clusteranalysis, frequency of use among expressed human antibodies, andavailable information on the crystal structures of human antibodies.Taking into account possible effects on antibody binding, VH-VL pairing,and other factors, murine residues were mutated to human residues wheremurine and human framework residues were different, with a fewexceptions. Additional humanization strategies were designed based on ananalysis of human germline antibody sequences, or a subgroup thereof,that possessed a high degree of homology, i.e., sequence similarity, tothe actual amino acid sequence of the murine antibody variable regions.

Homology modeling was used was to identify residues unique to the murineantibody sequences that are predicted to be critical to the structure ofthe antibody combining site (the CDRs). Homology modeling is acomputational method whereby approximate three dimensional coordinatesare generated for a protein. The source of initial coordinates andguidance for their further refinement is a second protein, the referenceprotein, for which the three dimensional coordinates are known and thesequence of which is related to the sequence of the first protein. Therelationship among the sequences of the two proteins is used to generatea correspondence between the reference protein and the protein for whichcoordinates are desired, the target protein. The primary sequences ofthe reference and target proteins are aligned with coordinates ofidentical portions of the two proteins transferred directly from thereference protein to the target protein. Coordinates for mismatchedportions of the two proteins, e.g. from residue mutations, insertions,or deletions, are constructed from generic structural templates andenergy refined to insure consistency with the already transferred modelcoordinates. This computational protein structure may be further refinedor employed directly in modeling studies. It should be clear from thisdescription that the quality of the model structure is determined by theaccuracy of the contention that the reference and target proteins arerelated and the precision with which the sequence alignment isconstructed.

For the murine sequences 5G1, 13C5 and 9C11, a combination of BLASTsearching and visual inspection was used to identify suitable referencestructures. Sequence identity of 25% between the reference and targetamino acid sequences is considered the minimum necessary to attempt ahomology modeling exercise. Sequence alignments were constructedmanually and model coordinates were generated with the program Jackal(see Petrey, D., Xiang, Z., Tang, C. L., Xie, L., Gimpelev, M., Mitros,T., Soto, C. S., Goldsmith-Fischman, S., Kernytsky, A., Schlessinger,A., et al. 2003. Using multiple structure alignments, fast modelbuilding, and energetic analysis in fold recognition and homologymodeling. Proteins 53 (Suppl. 6): 430-435).

The primary sequences of the murine and human framework regions of theselected antibodies share significant identity. Residue positions thatdiffer are candidates for inclusion of the murine residue in thehumanized sequence in order to retain the observed binding potency ofthe murine antibody. A list of framework residues that differ betweenthe human and murine sequences was constructed manually.

The likelihood that a given framework residue would impact the bindingproperties of the antibody depends on its proximity to the CDR residues.Therefore, using the model structures, the residues that differ betweenthe murine and human sequences were ranked according to their distancefrom any atom in the CDRs. Those residues that fell within 4.5 Å of anyCDR atom were identified as most important and were recommended to becandidates for retention of the murine residue in the humanized antibody(i.e. back mutation).

For humanization of the 5G1 variable regions, the general approachprovided in the present invention was followed. First, a molecular modelof the 5G1 variable regions was constructed with the aid of the computerprograms ABMOD and ENCAD (Levitt, M., J. Mol. Biol. 168: 595-620(1983)). Next, based on a homology search against human V and J segmentsequences, the VH segment 21/28 (Dersimonian, H., et al., J. Immunol.139: 2496-2501 (1987)) and the J segment JH4 (Ravetch, J. V., et al.,Cell 27: 583-591 (1981)) were selected to provide the frameworks for theHu5G1 heavy chain variable region. For the 5G1 light chain variableregion, the VL segment HF-21/28 (Chastagner, P., et al., Gene 101:305-306 (1991)) and the J segment JK4 (Hieter, P. A., et al., J. Biol.Chem. 257: 1516-1522 (1982)) were used. The identity of the frameworkamino acids between 5G1 VH and the acceptor human 21/28 and JH4 segmentswas 72%, while the identity between 5G1 VL and the acceptor humanHF21/28 and JK4 segments was 83%. At framework positions in which thecomputer model suggested significant contact with the CDRs, the aminoacids from the mouse V regions were substituted for the original humanframework amino acids. This was done at residues 48, 67, 68, 70, 72, 74and 97 of the heavy chain. For the light chain, replacement was made atresidue 50. Framework residues that occurred only rarely at theirrespective positions in the corresponding human V region subgroups werereplaced with human consensus amino acids at those positions. This wasdone at residues 44 and 76 of the heavy chain, and at residues 2, 15,41, 42, 44 and 51 of the light chain.

For humanization of the 13C5 variable regions, the general approachprovided in the present invention was followed. First, a molecular modelof the 13C5 variable regions was constructed with the aid of thecomputer programs ABMOD and ENCAD (Levitt, M., J. Mol. Biol. 168:595-620 (1983)). Next, based on a homology search against human V and Jsegment sequences, the VH segment M60 (Schroeder, Jr., H. W. and Wang,J. Y., Proc. Natl. Acad. Sci. USA 87: 6146-6150 (1990)) and the Jsegment JH4 (Ravetch, J. V., et al., Cell 27: 583-591 (1981)) wereselected to provide the frameworks for the Hu13C5 heavy chain variableregion. For the Hu13C5 light chain variable region, the VL segmentIII-3R (Manheimer-Lory, A., et al., J. Exp. Med. 174: 1639-1652 (1991))and the J segment JK4 (Hieter, P. A., et al., J. Biol. Chem. 257:1516-1522 (1982)) were used. The identity of the framework amino acidsbetween 13C5 VH and the acceptor human M60 and JH4 segments was 74%,while the identity between 13C5 VL and the acceptor human III-3R and JK4segments was 75%.

At framework positions in which the computer model suggested significantcontact with the CDRs, the amino acids from the mouse V regions weresubstituted for the original human framework amino acids. This was doneat residues 22, 49 and 71 for the light chain. Framework residues thatoccurred only rarely at their respective positions in the correspondinghuman V region subgroups were replaced with human consensus amino acidsat those positions. This was done at residues 10, 46, 83, 84, 86 and 87of the heavy chain, and at residues 62 and 73 of the light chain.

Amino acid sequences of VL and VH of humanized mAbs are shown in Table10.

TABLE 10 List of amino acid sequences of humanized mAbs SEQ ID SequenceNo. Protein region 123456789012345678901234567890 70 VH 5G1.1EVQLVQSGAEVKKPGASVKVSCKASGYTFT TYGVSWVRQAPGQGLEWIGEIYPGNYNTYYNEKFRGKATMTTDTSTSTAYMELRSLRSDD TAVYYCSRWRTSYFSDYGYFDYWGQGTTVT VSS 71VL 5G1.1 DVVMTQSPLSLPVTLGQPASISCRSSQSLV HSHGNTYLHWYQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCSQSTHVPYTFGGGTKVEIKR 72 VH 5G1.2EVQLVQSGAEVKKPGASVKVSCKASGYTFT TYGVSWVRQAPGQGLEWIGEIYPGNYNTYYNEKFRGKATLTADKSTSTAYMELSSLRSDD TAVYFCSRWRTSYFSDYGYFDYWGQGTTVT VSS 73VL 5G1.2 DVVMTQSPLSLPVTLGQPASISCRSSQSLV HSHGNTYLHWYQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCSQSTHVPYTFGGGTKVEIKR 74 VH 5G1.3EVQLVQSGAEVKKPGASVKVSCKASGYTFT TYGVSWVRQAPGQGLEWIGEIYPGNYNTYYNEKFRGKATLTADKSTSTAYMELSSLRSED TAVYYCSRWRTSYFSDYGYFDYWGQGTLVT VSS 75VL 5G1.3 DIVMTQSPLSLPVTPGQPASISCRSSQSLV HSHGNTYLHWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCSQSTHVPYTFGGGTKVEIK 76 VH 13C5.1EVTLKESGPVLVKPTETLTLTCTFSGFSLS TSDMGVDWIRQPPGKALEWLAHIWWDDVKRYNPALKSRLTISKDTSKSQVVLTMTNMDPV DTATYYCARTVSSGYTYYAMDYWGQGTTVT VSS 77VL 13C5.1 DIQMTQSPSSLSASVGDRVTITCRASQDIR NYLNWYQRKPGKVVKLLIYYTSKLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQ GNTLPLTFGGGTKVEIKR 78 VH 13C5.2EVTLKESGPVLVKPTETLTLTCTFSGFSLS TSDMGVDWIRQPPGKALEWLAHIWWDDVKRYNPALKSRLTISKDTSKSQVVIAMTNMDPV DTATYYCARTVSSGYIYYAMDYWGQGTTVT VSS 79VL 13C5.2 DIQMTQTPSSLSASVGDRVTISCRASQDIR NYLNWYQRKPGKVVKLLIFYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYFCQQ GNTLPLTFGGGTKVEIKR 80 VH 13C5.5EVTLRESGPGLVKPTQTLTLTCTLYGFSLS TSDMGVDWIRQPPGKGLEWLAHIWWDDVKRYNPALKSRLTISKDTSKNQVVLKLTSVDPV DTATYYCARTVSSGYIYYAMDYWGQGTLVT VSS 81VL 1305.5 DIQMTQSPSSLSASVGDRVTISCRASQDIR NYLNWYQQKPGKAPKLLIFYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQ GNTLPLTFGGGTKVEIK 82 VH 9C11.1EVQLVQSGAEVKKPGASVKVSCKASGYTFT SSWIHWVRQAPGQGLEWIGMIHPSDSETRLNQKFKDRATMTVDKSTSTAYMELSSLRSED TAVYYCASTATDFDYWGQGTTVTVSS 83 VL 9C11.1DVVLTQTPLSLPVTPGEPASISCRSTQTLL NSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQNNYLPLTFGAGTKLEIKR 84 VH 9C11.2EVQLVQSGAEVKKPGASVKVSCKASGYTFT SSWIHWVNQAPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSTSTAYMELSSLRSED TAVYYCASTATDFDYWGQGTTVTVSS 85 VL 9C11.2DVVLTQTPLSLPVTPGEPASISCRSTQTLL NSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQNNYLPLTFGAGTKLEIKR 90 VN 5G1.5QVQLVQSGAEVKKPGASVKVSCKASGYTFT TYGVSWVRQAPGQGLEWIGEIYPGNYNTYYNEKFRGKATLTADKSTSTAYMELSSLRSED TAVYYCSRWRTSYFSDYGYFDYWGQGTLVT VSS 91VL 5G1.5 DIVMTQSPLSLPVTPGQPASISCRSSQSLV HSHGNTYLHWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCSQSTHVPYTFGGGTKVEIK 80 VH 13C5.5L2EEVTLRESGPGLVKPTQTLTLTCTLYGFSLS TSDMGVDWIRQPPGKGLEWLAHIWWDDVKRYNPALKSRLTISKDTSKNQVVLKLTSVDPV DTATYYCARTVSSGYIYYAMDYWGQGTLVT VSS 92VL 13C5.5L2E DIQ MTQSPSSLSASVGDRVTISCRASQDIRNYL NWYQQKPGKAPKLLIFYTS MKPRGVPSRFS GSGSGTDYTLTISSLQPEDIATYYCQQGNT LPLTFGGGTKVEIK 80 VH 13C5.5L2FEVTLRESGPGLVKPTQTLTLTCTLYGFSLS TSDMGVDWIRQPPGKGLEWLAHIWWDDVKRYNPALKSRLTISKDTSKNQVVLKLTSVDPV DTATYYCARTVSSGYIYYAMDYWGQGTLVT VSS 93VL 13C5.5L3F DIQMTQSPSSLSASVGDRVTISCRASQDIRNYLNWYQQKPGKAPKLLIFYTSKLHSGVPS RFSGSGSGTDYTLTISSLQPEDIATYYCQQ G L T PPLTFGGGTKVEIK 80 VH 13C5.5L2EL3F EVTLRESGPGLVKPTQTLTLTCTLYGFSLSTSDMGVDWIRQPPGKGLEWLAHIWWDDVKR YNPALKSRLTISKLTSKNQVVLKLTSVDPVDTATYYCARTVSSGYIYYAMDYWGQGTLVT VSS 94 VL 13C5.5L2EL3FDIQMTQSPSSLSASVGDRVTISCRASQDIR NYLNWYQQKPGKAPKLLIFYTS MKPR GVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQ G L T P PLTFGGGTKVEIK

Example 2.2.2 Construction of Humanized Antibodies

In silico constructed humanized antibodies described above wereconstructed de novo using oligonucleotides. For each variable regioncDNA, 6 oligonucleotides of 60-80 nucleotides each were designed tooverlap each other by 20 nucleotides at the 5′ and/or 3′ end of eacholigonucleotide. In an annealing reaction, all 6 oligos were combined,boiled, and annealed in the presence of dNTPs. Then DNA polymerase I,Large (Klenow) fragment (New England Biolabs #M0210, Beverley, Mass.)was added to fill-in the approximately 40 bp gaps between theoverlapping oligonucleotides. PCR was then performed to amplify theentire variable region gene using two outermost primers containingoverhanging sequences complementary to the multiple cloning site in amodified pBOS vector (Mizushima, S. and Nagata, S., (1990) Nucleic acidsResearch Vol 18, No. 17)). The PCR products derived from each cDNAassembly were separated on an agarose gel and the band corresponding tothe predicted variable region cDNA size was excised and purified. Thevariable heavy region was inserted in-frame onto a cDNA fragmentencoding the human IgG1 constant region containing 2 hinge-region aminoacid mutations by homologous recombination in bacteria. These mutationsare a leucine to alanine change at position 234 (EU numbering) and aleucine to alanine change at position 235 (Lund et al., 1991, J.Immunol., 147:2657). The variable light chain region was insertedin-frame with the human kappa constant region by homologousrecombination. Bacterial colonies were isolated and plasmid DNAextracted; cDNA inserts were sequenced in their entirety. Correcthumanized heavy and light chains corresponding to each antibody wereco-transfected into COS cells to transiently produce full-lengthhumanized anti-human IL-13 antibodies. For 13C5, pBOS vectors containingthe 13C5 heavy chain grafted cDNA and the 13C5 light chain grafted cDNAwere co-transfected into COS cells. Cell supernatants containingrecombinant chimeric antibody were purified by Protein A Sepharosechromatography and bound antibody was eluted by addition of acid buffer.Antibodies were neutralized and dialyzed into PBS. Several humanizedantibodies are described in Table 10.

The ability of purified humanized antibodies to inhibit IL-13 activitywas determined using the A-549 bioassay as described in Examples 1.1.C.The binding affinities of the humanized antibodies to recombinant humanIL-13 were determined using surface plasmon resonance (Biacore®)measurement as described in Example 1.1.B. Table 11 shows IC₅₀ valuesfrom the A-549 bioassays and the affinity of the first six humanizedantibodies described in Table 10 for human IL-13 wt and variant.

TABLE 11 Neutralization potency and affinity of humanized anti IL-13mAbs. Potency (IC₅₀), M Affinity {grave over ( )}to hIL-13wt mAbhIL-13wt hIL-13v k_(on) (1/M · s) k_(off) (1/s) K_(D) (M) 5G1-Chim7.69E−11 6.92E−11 9.15E+05 3.82E−05 4.17E−11 5G1.1 2.90E−11 7.41E−117.86E+05 2.14E−05 2.72E−11 5G1.2 2.95E−11 5.53E−11 8.35E+05 8.81E−051.05E−10 5G1.5 1.14E−10 6.55E−11 8.69E+05 1.91E−05 2.20E−11 13C5-1.07E−10 3.70E−11 1.70E+06 9.65E−05 5.68E−11 Chim 13C5.1 8.68E−103.69E−10 6.68E+05 4.74E−04 7.10E−10 13C5.2 1.93E−10 1.30E−10 1.26E+061.23E−04 9.79E−11 13C5.5 1.24E−10 6.90E−11 2.51E+06 1.76E−04 7.01E−11

The CDR sequences of the humanized antibody 13C5.5 were further mutatedusing techniques known in the art, and three additional humanizedantibodies were generated. The ability of these additional humanizedantibodies to inhibit human, cynomolgus and rhesus IL-13 activity wasdetermined using the A-549 bioassay as described in Examples 1.1.C. Thebinding affinities of the additional humanized antibodies to recombinanthuman, cynomolgus and rhesus IL-13 were determined using surface plasmonresonance (Biacore®) measurement as described in Example 1.1.B. Inaddition to binding and inhibiting human IL-13, these three additionalantibodies showed enhanced affinity for cynomolgus and rhesus IL-13.Table 12 shows IC₅₀ values from the A-549 bioassays, and Table 13 showsthe affinity of the additional humanized antibodies to human, cynomolgusand rhesus IL-13.

TABLE 12 Neutralization potency of additional humanized anti IL-13 mAbsPotency (IC₅₀, nM) mAb Human IL-13 Cynomolgus IL-13 Rhesus IL-1313C5.5L2E 0.18 1.20 0.40 13C5.5L3F 0.15 0.46 0.14 13C5.5L2EL3F 0.12 0.480.26

TABLE 13 Binding affinity of additional humanized anti IL-13 mAbsAffinity (K_(D), nM) mAb Human IL-13 Cynomolgus IL-13 Rhesus IL-1313C5.5L2E 0.12 0.52 0.29 13C5.5L3F 0.24 0.19 0.11 13C5.5L2EL3F 0.25 0.320.13

Example 2.2.3 Characterization of Humanized Anti IL-13 Antibodies

We have isolated monoclonal antibodies that block IL-13 binding to bothIL-13Rα1 and IL-13Rα2. Both ELISA-based receptor binding assay and125-I-labeled IL-13 binding assay on cell surface demonstrated that13C5, both murine version and humanized version (i.e. 13C5.5), were ableto effective block IL-13 binding to both receptors. Antibodies in thesame lineage as 13C5, including 25C8 and 33C3, were also able to blockIL-13 binding to both receptors.

Example 2.2.3.a Humanized Anti IL-13 Antibodies Block Binding of IL-13to IL-13 Receptor

To determine the ability of humanized antibody 13C5.5 to block IL-13binding to IL-13 receptors (IL-13Rα1 and IL-13Rα2), an ELISA-basedreceptor binding assay was used. High-binding 96-well ELISA plates werecoated with 4 ug/ml of recombinant human IL-13Rα1/Fc or IL-13Rα2/Fc (R&DSystems) in 100 ul/well coating buffer (Carbonate-bicarbonate buffer,Pierce) at 4° C. After 16 hr, coating solution was removed by flickingplate contents in sink, and plates were washed and blocked 4 times withSuperblock Blocking Buffer (240 ul/well) (Pierce). Humanized anti-IL-13mAb 13c5.5 and control mAbs (1:4 serially diluted from 40 ug/ml, 50ul/well) and Biotin-IL-13 (50 ul/well, final concentrations of 5 nM forhIL-13Ra1/Fc, and 0.5 nM for hIL-13Ra2/Fc) were added and incubated for2 hr at room temperature (RT). Plates were washed 5 times with 300 ul0.1% PBST, and then 100 ul of 1:5000 diluted mouse anti-Biotin MAb(Jackson Immunosciences) was added and incubated at RT for 45 min. Theplates were washed again 5 times with 300 ul 0.1% PBST, followed byaddition of TMB substrate reagent (100 ul/well, Pharmingen); developedfor 5 min, and stopped by adding 50 ul of 2M H2SO4 (VWR). ODs at 450 nmwere determined by spectrophotometry. The results are shown in Table 14.

Additionally, the receptor blocking properties of the humanized mAbswere also assessed by cell surface-based receptor binding assay usingIL-13Rα2-transfected COS cells. Recombinant human IL-13 was labeled with¹²⁵I (Amersham, Arlington Heights, Ill.), using IODO-GEN reagent(Pierce, Rockford, Ill.) as previously described (Obiri N I et al.,(1995) J Biol Chem. 270:8797-8804). The specific activity of theradiolabeled IL-13 was estimated to be 158 μCi/μg protein. The labeledIL-13 exhibited similar bioactivity as unlabeled IL-13, as assessed bythe A-549 bioassay. For binding experiments, COS cells were transientlytransfected with human IL-13Ra2 by Lipofectamine 2000 (Invitrogen), andincubated for 48 hr. Transfected COS cells (5×105 cells in 100 μLbinding buffer: RPMI 1640 containing 0.2% human serum albumin and 10mmol HEPES) were incubated with 1.0 nM ¹²⁵I-IL-13 with or without 1 uMunlabeled IL-13 at 4° C. for 2 hours. Cell-bound ¹²⁵I-IL-13 wasseparated from unbound ¹²⁵I-IL-13 by centrifugation through a phthalateoil gradient, and radioactivity was determined with a gamma counter(Wallac, Gaithersburg, Md.). For antibody displacement assay,transfected COS cells were incubated with 125I-IL-13 (1.0 nM) with orwithout increasing concentrations (up to 50 ug/ml) of humanizedanti-IL-13 antibody 13C5.5, as described above. The results are shown inTable 14.

TABLE 14 Potency of mAbs in blocking human IL-13 (wt) binding to humanIL-13Rα2 in cell surface-based and ELISA-based receptor binding assaysPotency (IC₅₀, nM) mAb Cell surface ELISA 13C5.5  2.7  1.1 BAK502G9 75.834.3 5G1.5 P.B. P.B. mAb13.2 P.B. P.B. MJ2-7 17.6 19.0 P.B. Partialblockade that does not reach 50% inhibition.

Table 15 shows the binding affinity of the humanized 13C5.5 antibody andother anti-IL-13 antibodies.

TABLE 15 Binding affinity of anti-IL-13 mAbs as assessed by BiacoreAffinity (K_(D), nM) mAb Human IL-13 wt Human IL-13 variant 13C5.5 0.070.05 BAK502G9 2.10 0.17 mAb13.2 0.11 0.20 MJ2-7 1.14 0.79

In both cell surface-based and ELISA-based receptor binding assays,13C5.5 exhibits high potency in blocking human IL-13 binding to humanIL-13Rα2, with an IC₅₀ between 1 and 3 nM. While both BAK502G9 and MJ2-7were also able to reduce binding signal, their potencies were much lowerthan that of 13C5.5 (see Table 14), at least partially due to theirlower affinity for human wt IL-13 (see Table 15). MAb13.2 was not ableto inhibit IL-13 binding to IL-13Ra2, consistent with its epitope. Inaddition, 13C5.5 could achieve 100% inhibition in both assays at aconcentration of 100 nM (or 15 ug/ml). At the same concentration,BAK502G9 and MJ2-7 exhibited only 40% and 70% inhibition, respectively,in the cell surface-based receptor binding assay, and both exhibitedonly 60% inhibition in the ELISA-based receptor binding assay.

For a therapeutic mAb with serum half-life between 10 and 20 days inman, the serum concentration is normally between 5-15 ug/ml, with aweekly or bi-weekly IV or SC 3 mpk or less dosing regiment. Based onthis calculation, 13C5.5 is currently the only anti-IL-13 mAb that islikely to completely (100%) block human IL-13 binding to IL-13Rα2 invivo as a therapeutic mAb, at a serum concentration of 100 nM (or 15ug/ml), under a conventional dosing regimen of a monoclonal antibody.

Example 2.2.3.b Binding of Anti IL-13 Antibodies to Specific Epitope onIL-13

The epitopes on human IL-13 that the anti-IL-13 mAbs 13C5, 13C5.5, 9C11and 5G1 bind were mapped using an epitope excision technique, followedby peptide analysis with mass spectrometry (MS). In epitope excision,the protein was first bound to an immobilized mAb and then digested withproteolytic enzymes. Epitope regions on the protein were determined byusing MS and MS/MS to identify epitope-containing peptides.CNBr-activated Sepharose beads (Amersham Biosciences, 10 mg/reaction)were suspended in 500 uL of 0.1 M HCl and equilibrated for 15 min. Thebeads were transferred into compact reaction columns (USB Corporation)and washed with 0.1 M HCl followed by 0.1 M NaHCO3 coupling buffer. ThemAb (100 ug) was added to the suspension and incubated for 2 h with slowrotation at room temperature. Beads with the covalently attached mAbwere washed with 0.1 M Tris-HCl buffer ˜pH 8.0. Blocking of unreactedgroups on the CNBr Sepharose beads was accomplished by incubation for 2h with a 0.1 M Tris-HCl buffer ˜pH 8.0. Uncoupled mAb was removed bysequential washing with two buffers of different pH; 1) 0.1 M Naacetate, 0.5 M NaCl ˜pH 4.0 buffer and, 2) 0.1 M Tris-HCl, 0.5M NaCl ˜pH8.0 buffer. The beads were equilibrated in PBS ˜0.14 M NaCl, 2.7 mM KCl,4.3 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 and incubated for 2 h at roomtemperature, with and without IL-13. After washing the beads with PBS˜pH 7.2, an aliquot of the suspension was removed for MALDI-TOFanalysis.

The affinity bound protein was digested with different proteases(1:100˜1:20 enzyme:substrate ratio) for 12 h. Proteases used included:Trypsin, GluC, Chymotrypsin, Carboxypeptidase Y and Aminopeptidase M.Following proteolysis, the beads were washed with 500 uL of digestionbuffer. The last 100 uL of wash solution was saved as the control. About100 uL of 2% TFA was added to the beads and collected. Both the controland acid wash solutions were first concentrated to about 20 uL undervacuum. The peptides were then desalted with C18 ziptips. The sampleswere analyzed by MALDI-ToF MS, using either a Voyager DE or a VoyagerDE-Pro system. Analysis by nano-ESI-LC-MS/MS was performed on an Agilent1100 Capillary HPLC system interfaced to a Sciex Q-Star Pulsar i MSsystem.

In studying the epitopes of 13C5, two proteases used in sequential stepsgave the best results. With chymotrypsin, a major peptide consisting ofamino acid residues 100-130 of SEQ ID NO. 1 was detected, indicatingthat it may contain the epitope(s). Small amounts of peptides of aminoacid residues 103-130 and 104-130 of SEQ ID NO. 1 were also detected.Aminopeptidase M was used after the Chymotrypsin digestion. The majorpeptide detected was amino acid residues 104-130 of SEQ ID NO. 1,suggesting that the 4 N-terminal amino acid residues (80-83) were notpart of the epitope. Further digestion with carboxypeptidase Y resultedin loss of affinity. No peptide was observed after digestion andwashing. All peptide sequences were confirmed using nano-ESI-LC-MS/MS.

Epitope mapping of 13C5 and 13C5.5 indicated that its binding site(s)included the C-terminal Helix D region of human IL-13 (residues VRDTKIEVAQ FVKDL LL HLK KLFRE GR, corresponding to amino acid 104-130 of SEQID NO. 1). The c-terminal helix D region has been proposed to beinvolved in interactions with the IL-13 receptor (Zuegg et al 2001Immunol Cell Biol. 79:332-9).

Example 2.3 Crystallization of Anti-IL-13 Complexed to IL-13

The Fab portion of 13C5.5 was complexed with human IL-13 and crystals ofthe complex were generated as follows.

Example 2.3.1 Preparation and Purification of 13C5.5 Fab Fragment

To prepare 13C5.5 Fab fragment, 13C5.5 IgG in 0.15 M PBS buffer wasfirst concentrated to 2 mg/ml using an Ultrafree-15 Biomax 10 kDamolecular weight cut-off (MWCO) centrifugal filter device (Millipore).Papain gel slurry (Pierce) was pre-washed and charged in 2-3× withBuffer A (20 mM Na₂HPO₄, 10 mM EDTA, 20 mM cysteine) at a 1:1 volumeratio. The concentrated antibody was then mixed with 50% papain gelslurry and incubated at 37° C. for 24 hours with vigorous shaking. Theantibody/slurry mixture was centrifuged (Beckman 6KR) and thesupernatant was loaded onto a PBS pre-equilibrated Superdex 75. A majorpeak eluted and protein was pooled. A 25 mL Protein A Sepharose 4 FastFlow affinity column (Amersham Pharmacia) was prepared by washing with100 mL of PBS. The pooled antibody fragments were applied to theaffinity column (2 mL/min flow rate). Fractions containing 13C5.5 Fabfragments (monitored by UV absorbance at 280 nm) were collected in theflow-thru. Fractions containing a 13C5.5 Fab fragment concentrationgreater than 0.3 mg/mL (determined by UV absorbance at 280 nm) werepooled and frozen at −80° C. Sample purity was assessed with SDS-PAGE.

Example 2.3.2 IL-13/13C5.5 Fab Complex Preparation

Recombinant human IL-13 was expressed in a mammalian expression systemand subsequently purified using techniques well known in the art.Recombinant human IL-13 and 13C5.5 Fab protein were mixed at a 1:1 molarratio and incubated for 1 hour at 4° C. The complex sample was loadedonto a pre-equilibrated (20 mM Tris pH 7.5, 150 mM NaCl) Superdex 200column at 0.5 ml/min. Complex was pooled and concentrated to 24 mg/mLusing an Ultrafree-15 Biomax 10 kDa molecular weight cut-off (MWCO)centrifugal filter device (Millipore) and frozen at −80° C. Samplepurity was assessed with SDS-PAGE.

Example 2.3.3 Crystallization of IL-13/13C5.5 Fab Complex

Frozen IL-13/13C5.5 complex stock (˜24 mg/mL) was thawed on ice. Thecomplex (1.0 μL) was mixed with 1.0 μL of reservoir solution (1.75 MAmmonium Sulfate, 100 mM MES pH 6.5, 10 mM CaCl2). The resulting dropwas mixed in a sitting drop well (CrysChem sitting-drop plate) over thereservoir at about 18° C. Diamond-like crystals appeared within oneweek.

Example 2.3.4 Cryoprotection and Flash Cooling of IL-13/13C5.5 FabComplex Crystals

Crystals of IL-13/13C5.5 Fab complex were harvested using a fiber loopin mother liquor +20% glycerol. The crystals were subsequentlyflash-cooled by plunging into liquid nitrogen.

Example 2.3.5 X-ray Diffraction Data Collection of IL-13/13C5.5 FabComplex

X-ray diffraction data from IL-13/13C5.5 Fab crystals were collected atthe IMCA beamline at the Advanced Photon Source in Argonne, Ill. Thecrystals were maintained at a temperature of 100 K with an OxfordCryosystems Cryostream cooler during data collection. A total of 180frames were collected at an oscillation range of 1.0°. The data wereprocessed with the HKL2000 suite of programs (Otwinowski and Minor,1997). After determining the crystal orientation, the data wereintegrated with DENZO and scaled and merged with SCALEPACK, and placedon an absolute scale and reduced to structure factor amplitudes withTRUNCATE (French and Wilson, 1978). Five percent of the uniquereflections were assigned, in a random fashion, to the “free” set, forcalculation of the free R-factor (Rfree) (Brünger, 1992); the remaining95% of the reflections constituted the “working” set, for calculation ofthe R-factor (R). The x-ray diffraction data are summarized in Table 16.The following lists indexing for the crystal form: (1) IL-13/13C5.5 Fab:space group P2(1)2(1)2(1), a=163.578 Å, b=163.318 Å, c=228.627 Å,α=90.0°, β=90.0°, γ=90.0°. Table 17 lists the xray diffractionstatistics for the dataset.

TABLE 16 Summary of Crystallographic Unit Cell Information ofIL-13/13C5.5 Fab Complex a b c Crystal Space Group (Å) (Å) (Å) 1P2(1)2(1)2(1) 163.578 163.318 228.627

TABLE 17 Summary of X-ray Diffraction Data Statistics for IL-13/13C5.5Fab Complex. Resolution Unique Rsym Coverage Crystal Space Group (Å)Reflections (%)* (%)* Multiplicity* 1 P2(1)2(1)2(1) 47.1-2.60 188,9370.085 100.0 7.3 (0.562) (100.0) (7.3) *Highest resolution shell inparentheses.

Example 2.3.6 Molecular Replacement Solution and Refinement ofIL-13/13C5.5 Fab Complex Crystal Structure

A maximum likelihood molecular replacement solution was determined usingthe program PHASER (Read, 2001). A total of six 13C5.5 monomers weresolved at 3.0 Å resolution in the space group P2(1)2(1)2(1). The searchmodel was the crystal structure of Fab reported previously (Protein DataBank entry 1BJ1; Muller et al. 1998). Coordinates were generated basedon the molecular replacement solution.

The refinement of the IL-13/13C5.5 Fab complex crystal structure beganwith the molecular replacement solution coordinates, described above, inspace group P2(1)2(1)2(1). Refinement began using rigid-body refinementby the program REFMAC available in the CCP4 suite of programs (Murshudovet al., 1997, Collaborative Computational Project, 1994), which resultedin the following statistics at 2.6 Å: R of 40.00% (Rfree 39.00%). Denovo IL-13 electron density was observed. Manual building of six IL-13monomers was guided by the public IL-13 NMR structure 1IJZ (Moy et al.,2001) using the molecular graphics program O (Jones et al., 1991) andexamination of 2Fo-Fc and Fo-Fc electron-density maps. The refinementprogram REFMAC (Murshudov et al., 1997) was used for iterative rounds ofrestrained refinement resulting in the following statistics: R of 25.8%(Rfree 30.5%). Results are shown in Table 18.

TABLE 18 Summary of Crystallographic Refinement Statistics IL-13/13C5.5Fab Complex. Resolution Rfree R Crystal (Å) (%) (%) 1 10.0-1.50 30.525.8

Example 2.3.6 IL-13/13C5.5 Fab Complex Structure

Extensive contacts are observed between human IL-13 and multiple 13C5.5CDRs. The buried surface area at the antibody-antigen interface is1415.50 Å². The contacts are comprised of critical hydrogen bond andhydrophobic interactions that stabilize the interface. The two minimumsequence segments that comprise the majority of interface contacts areon IL-13 helices A and D (for structure of IL-13 see U.S. Patentpublication No. 2003-0013851 A1 incorporated herein by reference). Thesecontacts engage CDR's L1 and L3, and H2 and H3. Based on the foregoing,the epitope 13C5.5 binding range comprises the topographical regiondefined by Ser26-Asn38, Lys123-Arg130 of SEQ ID NO. 1. More preferably,the epitope 13C5.5 binding range comprises the topographical regiondefined by Arg30-Asn38, Lys123-Arg127 of SEQ ID NO. 1.

Example 2.4 In vivo Efficacy of Humanized IL-13 Antibodies

The in vivo efficacy of anti-hIL-13 antibodies was assessed as follows.

Example 2.4.1 In vivo Efficacy of Humanized IL-13 Antibodies in HumanIL-13 Induced Asthma Model

The efficacy of anti-hIL-13 antibodies 5G1, 13C5, and 13C5.5 were testedin a human IL-13 induced asthma model in mice. Mice were challenged withrecombinant human IL-13 at a dose of 1 μg in 50 μl sterile PBS,delivered into the trachea with a microsprayer using a rodentlaryngoscope to visualize the tracheal opening. A total of 2 doses ofIL-13 was given on days 1 and 2 of the study and airwayhyperresponsiveness (AHR; Hoymann, H. G.; J Pharmacol Toxicol Methods.2007 January-February; 55(1):16-26) as well as mucus, acidic mammalianchitinase (AMCase, Donnelly L E, Barnes P J., 1: Trends Pharmacol Sci.2004 October; 25(10):509-11) and thymus and activation regulatedchemokine (TARC; Bisset L R, Schmid-Grendelmeier P., Curr Opin Pulm Med.2005 January; 11(1):35-42) were measured in the broncho-alveolar lavagefluid 24 hr after the final challenge. Antibody doses of 100, 300, and1000 μg were administered by intra-peritoneal injection 1 day prior tothe first challenge with IL-13 and the results are summarized in Table19. 5G1 antibody, which does not block binding of IL-13 to eitherIL-13Rα1 or IL-13Rα2, was unable to neutralize IL-13 bioactivity in thisin vivo model with comparable levels of AHR, AMCase, and Muc5ac detectedin animals treated with 5G1 compared to PBS treated control animals. Incontrast, the 13C5 antibody that blocks binding to both α1 and α2receptors, was effective at reducing all parameters. Treatment withIL-13 increased airways resistance from 3.6 cm H₂O/ml/sec to 5.7 cmH₂O/ml/sec. Treatment with 13C5 (1000 μg) reduced airways resistance to4.3 cm H₂O/ml/sec. Mucus hyper-secretion as measured by muc5ac levelswere decreased from 356.5 units to a maximum 211 U with antibodytreatment corresponding to a 40% reduction. Similarly AMCase levels werereduced from 202 U to 68 U corresponding to a 66% reduction withcomparable reduction seen in TARC levels (n=10, p<0.05, all doses). Therecombinant humanized antibody 13C5.5 demonstrated similar results inthis model. IL-13 induced an increase in airways resistance followingchallenge with 30 μg/ml methacholine from 3.9 to 5.5 cm H₂O/ml/sec. Theantibody 13C5.5 inhibited airways resistance to 4.1, 4.45, and 4.3 cmH₂O/ml/sec at 100, 300 and 1000 μg doses respectively. Mucushyper-secretion as measured by muc5ac levels were reduced from 247 Uinduced by IL-13 treatment to 154, 30.2, and 11.1 U at 100, 300, and1000 μg doses of antibody treatment respectively. This represents a 38,88, and 96% inhibition of mucus production with this antibody. IL-13treatment induced 130 U AMCase activity that was reduced to 113, 98, and55 U by antibody treatment (100, 300, and 1000 μg doses) representing a14, 24, and 68% inhibition. These data demonstrate that 13C5 and therecombinant humanized antibody 13C5.5 that block binding of IL-13 toboth IL-13Rα1 and α2 can neutralize IL-13 induced responses of AHR,mucus, and AMCase production in the lung whereas antibodies that do notblock binding of IL-13 to α1 and α2 receptors are not effective atblocking all of these biological responses.

TABLE 19 Efficacy of anti-human IL-13 antibodies in IL-13 induced asthmamodel. AHR Mucus AMCase TARC Muc5ac arbitrary Resistance % Units % units% pg/ml % Antibody Dose (SEM) Inhibition (SEM) Inhibition (SEM)Inhibition (SEM) Inhibition 5G1 0 5.7 (0.38) -0- 258 (37.2) -0- 314.9(26.1) -0- 63.2 (14) -0- 100 315 (61) -0- 225.2 (17.1)  9 111 (34.5) -0-300 367.2 (63.2) -0- 277 (21.3) 12 94.1 (24.2) -0- 1000 5.3 (0.35)  7345.9 (61.6) -0- 255.1 (18.6) 19 90.2 (17.1) -0- 13C5 0 5.7 (0.38) 356.5(15.8) -0- 202.2 (18.8) -0- 91.7 (41.7) -0- 100 246 (30.6) 31 146.6(17.9)** 28 36 (10.3) 61 300 243.2 (36.7) 32 97.2 (10.8)** 52 23.3 (4.1)75 1000 4.3 (0.77)* 211.6 (28)*** 41 68.3 (9.2)*** 66 34.4 (12.1) 6213C5.5 0 5.54 (0.53) -0- 247.1 (96.4) -0- 130.4 (20.6) -0- NT 100 4.16(0.29)* 89 153.8 (67.6) 38 113.4 (18) 13 NT 300 4.45 (0.41)* 70 30.2(15.2)** 88 98.9 (10.6) 24 NT 1000 4.3 (0.27)* 79 11.1 (5.8)*** 96 55.5(6.4)*** 57 NT *p < 0.05, Student's T-test **p < 0.05, ANOVA,Bonferroni's ***p < 0.01, ANOVA, Bonferroni's

In another study, the efficacy of anti-hIL-13 antibodies BAK502G9, MJ2-7and 13C5.5 were compared in the human IL-13 induced asthma model inmice. Mice were challenged with recombinant human IL-13 at a dose of 1μg in 50 μl sterile PBS, delivered intranasally under light sedation. Atotal of 2 doses of IL-13 was given on days 1 and 2 of the study andairway hyperresponsiveness, as well as mucus, and AMCase, were measuredin the broncho-alveolar lavage fluid 24 hr after the final challenge.Antibody doses of 1000 μg were administered by intra-peritonealinjection 1 day prior to the first challenge with IL-13. Results of thestudy are summarized in Table 20. The 13C5.5 antibody, which blocksbinding of IL-13 to both IL-13α1 and α2 receptors, was effective atsignificantly reducing all parameters. Treatment with IL-13 increasedairways resistance following challenge with 30 mg/ml methacholine from4.2 cm H₂O/ml/sec to 7.2 cm H₂O/ml/sec. Treatment with 13C5.5 (1000 μg)reduced airways resistance by 86.8% to 4.6 cm H₂O/ml/sec. Mucushyper-secretion as measured by muc5ac levels were decreased from 768.2units to 412.9 U with antibody treatment corresponding to a 58.8%reduction. Similarly AMCase levels were reduced from 316.5 U to 147 Ucorresponding to a 52% reduction (n=10, p<0.001). Both the BAK502G9 andthe MJ2-7 antibodies, which inhibit IL-13 binding to IL-13Rα1 but do noteffectively inhibit IL-13 binding to IL-13Rα2 demonstrated comparableability to neutralize IL-13 induced AHR in this model. The antibodiesBAK502G9 and MJ2-7 only inhibited airways resistance from 7.2 to 5.96 cmH₂O/ml/sec and 5.93 cm H₂O/ml/sec respectively, representing a 42% and41.5% reduction in AHR. Mucus hyper-secretion as measured by muc5aclevels were reduced from 768.2 U induced by IL-13 treatment to 627.8 and380 U at 1000 μg doses of antibody corresponding to a 23% and 64%inhibition by BAK502G9 or the MJ2-7 antibodies respectively. TheBAK502G9 antibody was less effective at inhibiting AMCase compared toeither 13C5.5 or MJ2-7 antibodies. IL-13 treatment induced 316.5 UAMCase activity that was reduced to 279, and 169 U by either BAK502G9 orMJ2-7 antibody treatment (1000 μg dose) representing an 8% and 45%inhibition, respectively. These data demonstrate that the recombinanthumanized antibody 13C5.5 that blocks binding of IL-13 to both IL-13Rα1and α2 is most effective at neutralizing IL-13 induced responses of AHR,mucus, and AMCase production in the lung whereas antibodies that blockbinding of IL-13 to IL-13α2 receptor with lower affinity are not aseffective at blocking these biological responses that contribute to thepathogenesis of asthma.

TABLE 20 Comparison of 13C5.5, BAK502G9, and MJ2-7 antibodies in theIL-13 induced asthma model. Mucus AMCase AHR Muc5ac arbitrary DoseResistance % Units % units % Antibody (μg) (SEM) inhibition (SEM)inhibition (SEM) inhibition PBS 0 7.2 (0.77) -0- 768.2 (108)  -0- 316.5(43)   -0- 13C5.5 1000 4.6 (0.3)  86.8 412.9 (77.3) 46 147 (27) 54BAK502G9 1000 5.9 (0.38) 41.7 627.8 (59.7) 18 279.4 (28.5) 12 MJ2-7 10005.9 (0.67) 42.5   380 (48.5) 50.5 169 (20) 47

Example 2.4.2 In vivo Efficacy of IL-13 Antibodies in an OVA-inducedAsthma Mouse Model

To determine if receptor blockade properties (particularly regarding toIL-13Rα2) impact the vivo efficacy of the mAbs in asthma mouse models, apanel of rat anti-mouse IL-13 antibodies that exhibited differentreceptor blockade properties, as determined by receptor binding ELISAusing mIL-13Rα1/Fc and mIL-13Rα2/Fc proteins (R&D Systems) (see Table21) were generated. Since anti-hIL-13 mAb 3H7 cross-reacts with mouseIL-13, its anti-mIL-13 properties are also included in Table 21. Thebinding affinities of the antibodies for mouse IL-13 were measured usingBIACORE assay against recombinant mouse IL-13 (R&D Systems), andpotencies (IC50) of antibodies against mouse IL-13 were determined byA-549 bioassay against recombinant mouse IL-13. The variable domainsequences of 51D9 and 48D3 are shown in Table 22.

TABLE 21 Characterization of anti-mIL-13 monoclonal antibodies Blocksmouse IL-13 binding to mIL- mIL- Clone # Isotype K_(D) (M) IC₅₀ (M)13Rα1 13Rα2  3H7 Mouse IgG1 1.12E−08 2.43E−9  Yes Yes 51D9 Rat IgG1κ1.45E−10 3.43E−10 Yes Yes 48D3 Rat IgG1κ 1.05E−10 4.91E−11 Yes No 53F5Rat IgG1κ 2.82E−10 2.89E−10 No No 74H2 Rat IgG2aκ 3.92E−10 9.76E−10 NoNo 25C7 Rat IgG2aλ 4.22E−10 6.09E−10 Yes Yes 54D1 Rat IgG1κ 3.40E−112.40E−11 Yes Yes

TABLE 22 List of amino acid sequences of VH and VLregions of rat anti-mIL-13 mAbs SEQ ID Sequence No. Protein region123456789012345678901234567890 86 VH 51D9 QIQLVQSGPELKKPGESVKISCKASGYTFTDYAMHWVKQAPGKGLKWMAWINTYTGKPTY ADDFKGRFVFSLEASASTATLQISNLKNEDTATYFCARAGRTEGTHYYAMDAWGQGTSVT VSS 87 VL 51D9DIVLTQSPVLAVSLGQRATISCRASQSVSI SSSDLMHWYQQRPGHQPKLLIYRTSNLVSGIPARFSGSGSGTDFTLTIDPVQADDIAAYY CQQGRESPWTFGGGTKLELKR 88 VH 48D3EVQLVESGGDLVQPGRSLKLSCAASGFTFS DYYMAWVRQAPTKGLEWVASISNDGISTYYRDSVKGRFTISREKAKSSLYLQMDSLRSED TATYYCTTWNWEFGFFDYWGQGVMVTVSA 89 VL 48D3DIVLTQSPALAVSLGQRATISCRASQSVTI SRYNRMHWYQQRPGQQPKLLIYRSSYLASGIPARFSGSGSGTDFTLTIYPVQADDIATYY CQQNRESPWTFGGGTKLELNR

For the in vivo efficacy study in a murine asthma model, animals (femaleBalb/c mice) were purchased from Taconic, housed at Abbott BioresearchCenter, and utilized at 8-12 weeks of age. All protocols were approvedby the IACUC. Mice were sensitized to OVA (Sigma, endotoxin was removedfrom ovalbumin using DetoxiGel (Pierce) according to manufacturer'sprotocol and the final material contained less than 0.1 EU/mg protein)on day 0 and 7 with an intra peritoneal injection of 8 ug OVA in 2 mgalum (Pierce). On days 14 and 16, animals received intra-nasal challengeof 0.3 mg OVA in 50 ml sterile PBS. Antibodies 51D9 and 48D3 (purifiedfrom the supernatant of hybridoma clones 51D9 and 48D3 according tostandard procedures, which contained less than 0.1 EU/mg of protein andwere negative for rodent pathogens by PCR testing) were administered onday 13 as a single intra peritoneal injection in sterile PBS.Dexamethasone (Sigma) was administered orally once a day on days 13-17at a dose of 3 mg/kg. All endpoints were analyzed on day 17, 24 hr afterthe 2nd OVA challenge. Airway hyperresponsiveness (AHR) was assessedusing whole body plethysmography. Briefly, a surgical plane ofanesthesia was induced with an intra peritoneal injection of ketamineand xylazine. A tracheal canula was surgically inserted between the 3rdand 4th tracheal rings. Spontaneous breathing was prevented by intravenous jugular injection of pancuronium bromide and animals were placedin a whole body plethysmograph (Buxco) and mechanically ventilated with0.2 ml room air at 150 breaths per minute with a volume controlledventilator (Harvard Apparatus). Pressure in the lung and flow within theplethysmograph were measured using transducers and lung resistance wascalculated as pressure/flow using Biosystem Xa software. Baselineresistance as well as resistance following challenge with methacholine(3, 10, & 30 mg/ml) that was delivered with an inline ultrasonicnebulizer were measured. Upon completion of pulmonary function testing,the lungs were lavaged 4 times with 0.5 ml sterile PBS. Lavage fluid wasanalyzed for TARC, AMCase and cellular infiltrate. Serum was collectedfor quantification of antibody levels at the conclusion of the study.

Murine TARC levels were determined by ELISA (R&D) according tomanufacturer's protocol. AMCase activity was determined inbronchoalveolar lavage (BAL) fluid (1 to 10 dilution with 0.01% BSA, 30mM sodium citrate, 60 mM sodium phosphate, pH 5.2 in the presence of 80uM 4-methylumbelliferyl β-D-N,N′-diacetylchitobioside. Reactions wereincubated for 15 minutes at room temperature and quenched by addition of100 uL of 1 M glycine/NaOH pH 10.6. Product formation was determined byfluorescence emission at 460 nm using excitation at 385 nm on aFluoroskan Ascent fluorometer. To assess goblet cell hyperplasia, lungswere inflated with 10% neutral buffered formalin at 22 cm height for 15minutes to achieve consistent area of lung surface. Sections wereembedded in paraffin, sectioned, and stained with periodic acid schiff(PAS). The area of PAS positive cells along the main bronchus of theleft lung was quantitated using ImagePro Plus Software. Muc5ac levelswere determined by ELISA. A 96 well plate is coated with BAL fluid,dried overnight, and then a biotinylated anti-Muc5ac antibody is addedand detected with HRP conjugated streptavidin, followed by cleavage ofcolorimetric substrate TMB.

The relative contribution of IL-13Rα1 and α2 towards the pathogenesis ofasthma was tested in a standard model of ovalbumin-induced asthma inmice. Antibodies that blocked binding of IL-13 to both α1 and α2receptor (51D9, 54D1, and 3H7 with potencies of 340, 24, and 2430 pMrespectively) as well as an antibody that blocked binding of IL-13 toonly α1 receptor (48D3, potency of 50 pM) were tested by treatinganimals with the antibodies one day prior to the local challenges withovalbumin and the results are presented in Table 23. OVA challengeinduced increases in lung resistance following challenge withmethacholine, mucus hypersecretion as measured by increased levels ofMuc5ac in the BAL fluid as well as increased PAS positive staining ofepithelial cells by histological assessment, infiltration of the lungwith eosinophils & T cells, and production of asthma-related proteinsAMCase and TARC.

Antibodies that blocked the binding of IL-13 to both IL-13 Rα1 and α2all demonstrated efficacy and the in vivo potency of the reagentsshifted in accordance with their measured potency in vitro. 51D9 wastested at doses of 100, 300, and 1000 ug/mouse. OVA treatment caused anincrease in airways resistance following challenge with 30 mg/mlmethacholine to 6.2 cm H₂O/ml/sec compared to 3.6 cm H₂O/ml/sec innon-asthmatic animals. Treatment of mice with 51D9 completely preventedthe increase in lung resistance with values comparable to that observedin non-asthmatic control animals of 4.1, 4.0, and 3.5 cm H₂O/ml/sec fordoses of 100, 300, and 1000 μg respectively (n=8-10/group; p<0.05). Theamount of inhibition observed with 51D9 was comparable to that achievedwith steroid treatment (3.3 cm H₂O/ml/sec). Treatment with 51D9 alsodose dependently inhibited mucus hypersecretion from 404 units Muc5acdown to 55 U in animals treated with 1000 μg of 51D9. Inhibition ofmucus hyper-secretion was also observed by histological assessment ofPAS reactive epithelial cells. The area of percent positive cells wasdecreased from 1.0% to 0.6% and 0.5% with 300 and 1000 ug dose of 51D9antibody respectively representing a decrease of 47-65% (n=8, p<0.01).51D9 treatment also inhibited TARC and AMCase. OVA challenge induced 61pg/ml of TARC that was reduced to 7.8 pg/ml with 1000 ug 51D9 treatment(n=10, p<0.05). OVA challenge induced 96 arbitrary units of AMCaseactivity that was dose dependently reduced with 51D9 to 52, 45 and 21 Uwith 100, 300 and 1000 μg of antibody respectively (n=9-10, p<0.01).54D1 which has a 10 fold greater in vitro potency (24 pM) demonstratedinhibition of AHR at the 30 ug dose with reduction of airways resistancefrom 6.58 cm H₂O/ml/sec to 4.4 cm H₂O/ml/sec and maximal inhibitionobserved with treatment with 300 ug of antibody to reduce airwaysresistance to a value of 3.65 cm H₂O/ml/sec. Similar potency wasobserved at inhibition of mucus, AMCase and TARC production. A thirdantibody 3H7, which has a potency of 2.5 nM, still demonstratedinhibition of AHR, mucus, and AMCase levels but only at a dose of 1000μg antibody treatment consistent with a 10 fold shift in potencydescribed in in vitro bioassays.

The efficacy of antibodies that block binding of IL-13 to only IL-13Rα1was tested with the antibody 48D3. Animals were treated with of 30, 100,300, and 1000 μg/mouse. OVA challenge induced a rise in airwaysresistance to 5.69 cm H20/ml/sec compared to 4.1 cm H20/ml/sec innon-asthmatic PBS treated animals. Treatment with 30 μg 48D3 had noeffect on OVA-induced lung resistance while treatment with 100, 300 and1000 ug 48D3 inhibited OVA-induced changes in lung resistance to amaximum of 4.4 cm H₂O/ml/sec or to ˜80% of the OVA control levels (n=10,p<0.05). In contrast to effects observed with 51D9, 48D3 did not inhibitOVA-induced mucus hypersecretion as measured by either Muc5ac ELISA orPAS reactive epithelial cells. A slight but statistical significantreduction in mucus (30%) was observed with 48D3 treatment at the 30 μgdose, whereas all other doses were equivalent to OVA challenged animals.Histological quantitation of PAS positive staining demonstrated 0.6% inOVA challenged animals vs 0.8% in animals challenged with OVA andtreated with 1000 ug 48D3. In these studies 48D3 inhibited OVA-inducedAMCase expression from 196 U detected in OVA-treated animals to 63, 90,87, and 96 U at 30, 100, 300 and 1000 ug doses respectively. Analysis ofantibody levels indicated that comparable levels of 48D3 and 51D9 weredetectable in both the serum and BAL fluid of antibody treated mice.Despite the 7 fold greater potency of 48D3 antibody compared to the 51D9antibody, and equivalent exposure of the two antibodies, the 48D3antibody was not able to inhibit AHR or AMCase to the same extent as theantibody that blocked binding of IL-13 to IL-13Rα1 and α2 and was unableto attenuate mucus production. Together these data suggest that IL-13Rα2plays a central role in mediating OVA-induced mucus hyper-secretion andthat IL-13Rα2 contributes towards regulating the asthmatic phenotype invivo.

TABLE 23 Efficacy of anti-mouse IL-13 antibodies in murine model ofovalbumin-induced asthma. AHR Mucus AMCase TARC % Muc5ac % arbitrary % %Antibody Dose Resistance Inhibition Units Inhibition units Inhibitionpg/ml Inhibition 54D1 0 6.585 (.89) -0- 573 (96.2) -0- 112.1 (19.3) -0-141 (43.2) -0- 30 4.486 (0.3)* 34 203 (22)* 65 30.8 (4.8)* 72.5 38.3(10.6)* 63 100 4.2 (0.32)* 37 153 (44)* 74 14.4 (2.7)* 87.3 23.8 (7.3)*83 300 3.65 (0.22)* 45 77.3 (6.9)* 87 11.0 (1.5)* 90.2 17.2 (4.5)* 881000 3.58 (0.34)* 46 79 (8.5)* 87 10.4 (1.2)* 90.1 20 (12.3)* 86 51D9 06.24 (1.4) -0- 409.2 (36.4) -0- 97.6 (11) -0- 61.8 (12.1)* -0- 100 4.13(0.91)* 33.8 188.8 (24.8)* 54 52.9 (10.9)* 46 25.2 (12.8)* 60 300 4.06(0.32)* 34.8 180.8 (32.4)* 56 45.8 (13.7)* 53 23.1 (12.9)* 62 1000 3.57(0.78)* 43 55.1 (23.4)* 87 21.2 (7.8)* 79 7.8 (4.2)* 87 3H7 0 7.9 (1.2)-0- 965 (59.9) -0- 129.7 (17.2) -0- 173.9 (32.0) -0- 1000 6.02 (0.31)*24 587 (48.4)* 40 78.18 (12.3)* 40 77.3 (18.5)* 56 48D3 0 5.69 (0.42)-0- 666.7 (74.7) -0- 196.6 (35.5) -0- NT 30 5.288 (0.43) 8.1 445.4(33.8)* 34 63.1 (18.2)* 68 NT 100 4.5 (0.42)* 19.5 567.5 (62.6) 15 90.4(15.4)* 54 NT 300 5.25 (0.42) 14.1 606.3 (71.2)  9 87.4 (19.6)* 55 NT1000 4.4 (0.33)* 20.7 534.9 (31) 20 96.5 (10.4)* 51 NT *denotes p < .05,ANOVA

We claim:
 1. An isolated nucleic acid encoding an amino acid sequence ofat least one variable domain of an IL-13 binding protein, wherein saidat least one variable domain comprises CDR-H1, CDR-H2, and CDR-H3 aminoacid sequences independently selected from the group consisting of:CDR-H1 CDR-H2 CDR-H3 residues 31-35 of residues 50-66 of residues 99-105of SEQ ID NO.: 32 SEQ ID NO.: 32 SEQ ID NO.: 32 residues 31-35 ofresidues 50-66 of residues 99-105 of SEQ ID NO.: 34 SEQ ID NO.: 34 SEQID NO.: 34 residues 31-35 of residues 50-66 of residues 99-109 of SEQ IDNO.: 36 SEQ ID NO.: 36 SEQ ID NO.: 36 residues 31-35 of residues 50-66of residues 99-109 of SEQ ID NO.: 38 SEQ ID NO.: 38 SEQ ID NO.: 38residues 31-35 of residues 50-66 of residues 99-112 of SEQ ID NO.: 39SEQ ID NO.: 39 SEQ ID NO.: 39 residues 31-35 of residues 50-66 ofresidues 99-112 of SEQ ID NO.: 41 SEQ ID NO.: 41 SEQ ID NO.: 41 residues31-35 of residues 50-66 of residues 99-100 of SEQ ID NO.: 42 SEQ ID NO.:42 SEQ ID NO.: 42 residues 31-35 of residues 50-65 of residues 98-106 ofSEQ ID NO.: 44 SEQ ID NO.: 44 SEQ ID NO.: 44 residues 31-37 of residues52-67 of residues 100-112 of SEQ ID NO.: 46 SEQ ID NO.: 46 SEQ ID NO.:46 residues 31-37 of residues 52-67 of residues 100-112 of SEQ ID NO.:48 SEQ ID NO.: 48 SEQ ID NO.: 48 residues 31-37 of residues 52-67 ofresidues 100-112 of SEQ ID NO.: 50 SEQ ID NO.: 50 SEQ ID NO.: 50residues 31-35 of residues 50-66 of residues 99-107 of SEQ ID NO.: 52SEQ ID NO.: 52 SEQ ID NO.: 52 residues 31-35 of residues 50-65 ofresidues 98-107 of SEQ ID NO.: 54 SEQ ID NO.: 54 SEQ ID NO.: 54 residues31-35 of residues 50-65 of residues 98-107 of SEQ ID NO.: 56 SEQ ID NO.:56 SEQ ID NO.: 56 residues 31-35 of residues 50-65 of residues 98-107 ofSEQ ID NO.: 58 SEQ ID NO.: 58 SEQ ID NO.: 58 residues 31-35 of residues50-65 of residues 98-107 of SEQ ID NO.: 60 SEQ ID NO.: 60 SEQ ID NO.: 60residues 31-35 of residues 50-65 of residues 98-107 of SEQ ID NO.: 62SEQ ID NO.: 62 SEQ ID NO.:
 62.


2. An isolated nucleic acid encoding an amino acid sequence of at leastone variable domain of an IL-13 binding protein, wherein said at leastone variable domain comprises CDR-L1, CDR-L2, and CDR-L3 amino acidsequences independently selected from the group consisting of: CDR-L1CDR-L2 CDR-L3 residues 24-39 of SEQ ID NO.: 33 residues 55-61 of SEQ IDNO.: 33 residues 94-102 of SEQ ID NO.: 33 residues 24-39 of SEQ ID NO.:35 residues 55-61 of SEQ ID NO.: 35 residues 94-102 of SEQ ID NO.: 35residues 24-39 of SEQ ID NO.: 37 residues 55-61 of SEQ ID NO.: 37residues 94-102 of SEQ ID NO.: 37 residues 24-39 of SEQ ID NO.: 40residues 55-61 of SEQ ID NO.: 40 residues 94-102 of SEQ ID NO.: 40residues 24-39 of SEQ ID NO.: 43 residues 55-61 of SEQ ID NO.: 43residues 94-102 of SEQ ID NO.: 43 residues 24-40 of SEQ ID NO.: 45residues 56-62 of SEQ ID NO.: 45 residues 95-103 of SEQ ID NO.: 45residues 24-34 of SEQ ID NO.: 47 residues 50-56 of SEQ ID NO.: 47residues 89-97 of SEQ ID NO.: 47 residues 24-34 of SEQ ID NO.: 49residues 50-56 of SEQ ID NO.: 49 residues 89-97 of SEQ ID NO.: 49residues 24-34 of SEQ ID NO.: 51 residues 50-56 of SEQ ID NO.: 51residues 89-97 of SEQ ID NO.: 51 residues 23-36 of SEQ ID NO.: 53residues 52-58 of SEQ ID NO.: 53 residues 91-99 of SEQ ID NO.: 53residues 24-38 of SEQ ID NO.: 55 residues 54-60 of SEQ ID NO.: 55residues 93-101 of SEQ ID NO.: 55 residues 24-38 of SEQ ID NO.: 57residues 54-60 of SEQ ID NO.: 57 residues 93-101 of SEQ ID NO.: 57residues 24-38 of SEQ ID NO.: 59 residues 54-60 of SEQ ID NO.: 59residues 93-101 of SEQ ID NO.: 59 residues 24-38 of SEQ ID NO.: 61residues 54-60 of SEQ ID NO.: 61 residues 93-101 of SEQ ID NO.: 61residues 24-38 of SEQ ID NO.: 63 residues 54-60 of SEQ ID NO.: 63residues 93-101 of SEQ ID NO.: 63 residues 24-34 of SEQ ID NO: 92residues 50-56 of SEQ ID NO: 92 residues 89-97 of SEQ ID NO: 92 residues24-34 of SEQ ID NO: 93 residues 50-56 of SEQ ID NO: 93 residues 89-97 ofSEQ ID NO: 93 residues 24-34 of SEQ ID NO: 94 residues 50-56 of SEQ IDNO: 94 residues 89-97 of SEQ ID NO:
 94.


3. The isolated nucleic acid according to either claim 1 or claim 2,encoding an antibody construct amino acid sequence, wherein saidantibody construct comprises said binding protein and further comprisesa linker polypeptide or an immunoglobulin constant domain.
 4. A vectorcomprising an isolated nucleic acid according to claim 1 or an isolatednucleic acid according to claim 2, or a combination thereof.
 5. Thevector of claim 4 wherein said vector is selected from the groupconsisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ.
 6. A hostcell comprising a vector according to claim
 4. 7. The host cellaccording to claim 6, wherein said host cell is a prokaryotic cell. 8.The host cell according to claim 7, wherein said host cell is E. coli.9. The host cell according to claim 6, wherein said host cell is aeukaryotic cell.
 10. The host cell according to claim 9, wherein saideukaryotic cell is selected from the group consisting of a protist cell,an animal cell, a plant cell, and a fungal cell.
 11. The host cellaccording to claim 9, wherein said eukaryotic cell is an animal cellselected from the group consisting of: a mammalian cell, an avian cell,and an insect cell.
 12. The host cell according to claim 9, wherein saidhost cell is a CHO cell.
 13. The host cell according to claim 9, whereinsaid host cell is a COS cell.
 14. The host cell according to claim 9,wherein said host cell is a yeast cell.
 15. The host cell according toclaim 14, wherein said yeast cell is Saccharomyces cerevisiae.
 16. Thehost cell according to claim 9, wherein said host cell is an insect Sf9cell.
 17. A host cell comprising at least one nucleic acid according toeither claim 1 or claim
 2. 18. A host cell comprising one or morenucleic acids encoding a polypeptide chain of an IL-13 binding protein,said polypeptide chain comprising CDR-H1, CDR-H2, and CDR-H3 amino acidsequences independently selected from the group consisting of: CDR-H1CDR-H2 CDR-H3 residues 31-35 of SEQ ID NO.: 32 residues 50-66 of SEQ IDNO.: 32 residues 99-105 of SEQ ID NO.: 32 residues 31-35 of SEQ ID NO.:34 residues 50-66 of SEQ ID NO.: 34 residues 99-105 of SEQ ID NO.: 34residues 31-35 of SEQ ID NO.: 36 residues 50-66 of SEQ ID NO.: 36residues 99-109 of SEQ ID NO.: 36 residues 31-35 of SEQ ID NO.: 38residues 50-66 of SEQ ID NO.: 38 residues 99-109 of SEQ ID NO.: 38residues 31-35 of SEQ ID NO.: 39 residues 50-66 of SEQ ID NO.: 39residues 99-112 of SEQ ID NO.: 39 residues 31-35 of SEQ ID NO.: 41residues 50-66 of SEQ ID NO.: 41 residues 99-112 of SEQ ID NO.: 41residues 31-35 of SEQ ID NO.: 42 residues 50-66 of SEQ ID NO.: 42residues 99-100 of SEQ ID NO.: 42 residues 31-35 of SEQ ID NO.: 44residues 50-65 of SEQ ID NO.: 44 residues 98-106 of SEQ ID NO.: 44residues 31-37 of SEQ ID NO.: 46 residues 52-67 of SEQ ID NO.: 46residues 100-112 of SEQ ID NO.: 46 residues 31-37 of SEQ ID NO.: 48residues 52-67 of SEQ ID NO.: 48 residues 100-112 of SEQ ID NO.: 48residues 31-37 of SEQ ID NO.: 50 residues 52-67 of SEQ ID NO.: 50residues 100-112 of SEQ ID NO.: 50 residues 31-35 of SEQ ID NO.: 52residues 50-66 of SEQ ID NO.: 52 residues 99-107 of SEQ ID NO.: 52residues 31-35 of SEQ ID NO.: 54 residues 50-65 of SEQ ID NO.: 54residues 98-107 of SEQ ID NO.: 54 residues 31-35 of SEQ ID NO.: 56residues 50-65 of SEQ ID NO.: 56 residues 98-107 of SEQ ID NO.: 56residues 31-35 of SEQ ID NO.: 58 residues 50-65 of SEQ ID NO.: 58residues 98-107 of SEQ ID NO.: 58 residues 31-35 of SEQ ID NO.: 60residues 50-65 of SEQ ID NO.: 60 residues 98-107 of SEQ ID NO.: 60residues 31-35 of SEQ ID NO.: 62 residues 50-65 of SEQ ID NO.: 62residues 98-107 of SEQ ID NO.: 62

and nucleic acids encoding a polypeptide chain of an IL-13 bindingprotein, said polypeptide chain comprising CDR-L1, CDR-L2, and CDR-L3amino acid sequences independently selected from the group consistingof: CDR-L1 CDR-L2 CDR-L3 residues 24-39 of SEQ ID NO.: 33 residues 55-61of SEQ ID NO.: 33 residues 94-102 of SEQ ID NO.: 33 residues 24-39 ofSEQ ID NO.: 35 residues 55-61 of SEQ ID NO.: 35 residues 94-102 of SEQID NO.: 35 residues 24-39 of SEQ ID NO.: 37 residues 55-61 of SEQ IDNO.: 37 residues 94-102 of SEQ ID NO.: 37 residues 24-39 of SEQ ID NO.:40 residues 55-61 of SEQ ID NO.: 40 residues 94-102 of SEQ ID NO.: 40residues 24-39 of SEQ ID NO.: 43 residues 55-61 of SEQ ID NO.: 43residues 94-102 of SEQ ID NO.: 43 residues 24-40 of SEQ ID NO.: 45residues 56-62 of SEQ ID NO.: 45 residues 95-103 of SEQ ID NO.: 45residues 24-34 of SEQ ID NO.: 47 residues 50-56 of SEQ ID NO.: 47residues 89-97 of SEQ ID NO.: 47 residues 24-34 of SEQ ID NO.: 49residues 50-56 of SEQ ID NO.: 49 residues 89-97 of SEQ ID NO.: 49residues 24-34 of SEQ ID NO.: 51 residues 50-56 of SEQ ID NO.: 51residues 89-97 of SEQ ID NO.: 51 residues 23-36 of SEQ ID NO.: 53residues 52-58 of SEQ ID NO.: 53 residues 91-99 of SEQ ID NO.: 53residues 24-38 of SEQ ID NO.: 55 residues 54-60 of SEQ ID NO.: 55residues 93-101 of SEQ ID NO.: 55 residues 24-38 of SEQ ID NO.: 57residues 54-60 of SEQ ID NO.: 57 residues 93-101 of SEQ ID NO.: 57residues 24-38 of SEQ ID NO.: 59 residues 54-60 of SEQ ID NO.: 59residues 93-101 of SEQ ID NO.: 59 residues 24-38 of SEQ ID NO.: 61residues 54-60 of SEQ ID NO.: 61 residues 93-101 of SEQ ID NO.: 61residues 24-38 of SEQ ID NO.: 63 residues 54-60 of SEQ ID NO.: 63residues 93-101 of SEQ ID NO.: 63 residues 24-34 of SEQ ID NO: 92residues 50-56 of SEQ ID NO: 92 residues 89-97 of SEQ ID NO: 92 residues24-34 of SEQ ID NO: 93 residues 50-56 of SEQ ID NO: 93 residues 89-97 ofSEQ ID NO: 93 residues 24-34 of SEQ ID NO: 94 residues 50-56 of SEQ IDNO: 94 residues 89-97 of SEQ ID NO:
 94.


19. A method of producing a protein capable of binding IL-13, comprisingculturing the host cell of claim 6 in culture medium under conditionssufficient for expression of said binding protein.
 20. A host cellcomprising a first nucleic acid encoding a variable heavy chain (VH) setof 3 CDRs and a second nucleic acid encoding a variable light chain (VL)set of 3 CDRs, wherein said VH set of 3 CDRs is selected from any one ofthe VH sets in the group below and said VL set of 3 CDRs is selectedfrom any one of the VL sets in the group below: VH 25C8 CDR Set VH 25C8CDR-H1 Residues 31-35 of SEQ ID NO.: 32 VH 25C8 CDR-H2 Residues 50-66 ofSEQ ID NO.: 32 VH 25C8 CDR-H3 Residues 99-105 of SEQ ID NO.: 32 VL 25C8CDR Set VL 25C8 CDR-L1 Residues 24-39 of SEQ ID NO.: 33 VL 25C8 CDR-L2Residues 55-61 of SEQ ID NO.: 33 VL 25C8 CDR-L3 Residues 94-102 of SEQID NO.: 33 VH 9C11 CDR Set VH 9C11 CDR-H1 Residues 31-35 of SEQ ID NO.:34 VH 9C11 CDR-H2 Residues 50-66 of SEQ ID NO.: 34 VH 9C11 CDR-H3Residues 99-105 of SEQ ID NO.: 34 VL 9C11 CDR Set VL 9C11 CDR-L1Residues 24-39 of SEQ ID NO.: 35 VL 9C11 CDR-L2 Residues 55-61 of SEQ IDNO.: 35 VL 9C11 CDR-L3 Residues 94-102 of SEQ ID NO.: 35 VH 21D9 CDR SetVH 21D9 CDR-H1 Residues 31-35 of SEQ ID NO.: 36 VH 21D9 CDR-H2 Residues50-66 of SEQ ID NO.: 36 VH 21D9 CDR-H3 Residues 99-109 of SEQ ID NO.: 36VL 21D9 CDR Set VL 21D9 CDR-L1 Residues 24-39 of SEQ ID NO.: 37 VL 21D9CDR-L2 Residues 55-61 of SEQ ID NO.: 37 VL 21D9 CDR-L3 Residues 94-102of SEQ ID NO.: 37 VH 22D10 CDR Set VH 22D10 CDR-H1 Residues 31-35 of SEQID NO.: 38 VH 22D10 CDR-H2 Residues 50-66 of SEQ ID NO.: 38 VH 22D10CDR-H3 Residues 99-109 of SEQ ID NO.: 38 VL 22D10 CDR Set VL 22D10CDR-L1 Residues 24-39 of SEQ ID NO.: 37 VL 22D10 CDR-L2 Residues 55-61of SEQ ID NO.: 37 VL 22D10 CDR-L3 Residues 94-102 of SEQ ID NO.: 37 VH5F1 CDR Set VH 5F1 CDR-H1 Residues 31-35 of SEQ ID NO.: 39 VH 5F1 CDR-H2Residues 50-66 of SEQ ID NO.: 39 VH 5F1 CDR-H3 Residues 99-112 of SEQ IDNO.: 39 VL 5F1 CDR Set VL 5F1 CDR-L1 Residues 24-39 of SEQ ID NO.: 40 VL5F1 CDR-L2 Residues 55-61 of SEQ ID NO.: 40 VL 5F1 CDR-L3 Residues94-102 of SEQ ID NO.: 40 VH 5G1 CDR Set VH 5G1 CDR-H1 Residues 31-35 ofSEQ ID NO.: 41 VH 5G1 CDR-H2 Residues 50-66 of SEQ ID NO.: 41 VH 5G1CDR-H3 Residues 99-112 of SEQ ID NO.: 41 VL 5G1 CDR Set VL 5G1 CDR-L1Residues 24-39 of SEQ ID NO.: 40 VL 5G1 CDR-L2 Residues 55-61 of SEQ IDNO.: 40 VL 5G1 CDR-L3 Residues 94-102 of SEQ ID NO.: 40 VH 3H7 CDR SetVH 3H7 CDR-H1 Residues 31-35 of SEQ ID NO.: 42 VH 3H7 CDR-H2 Residues50-66 of SEQ ID NO.: 42 VH 3H7 CDR-H3 Residues 99-100 of SEQ ID NO.: 42VL 3H7 CDR Set VL 3H7 CDR-L1 Residues 24-39 of SEQ ID NO.: 43 VL 3H7CDR-L2 Residues 55-61 of SEQ ID NO.: 43 VL 3H7 CDR-L3 Residues 94-102 ofSEQ ID NO.: 43 VH 14B2 CDR Set VH 14B2 CDR-H1 Residues 31-35 of SEQ IDNO.: 44 VH 14B2 CDR-H2 Residues 50-65 of SEQ ID NO.: 44 VH 14B2 CDR-H3Residues 98-106 of SEQ ID NO.: 44 VL 14B2 CDR Set VL 14B2 CDR-L1Residues 24-40 of SEQ ID NO.: 45 VL 14B2 CDR-L2 Residues 56-62 of SEQ IDNO.: 45 VL 14B2 CDR-L3 Residues 95-103 of SEQ ID NO.: 45 VH 13C5 CDR SetVH 13C5 CDR-H1 Residues 32-38 of SEQ ID NO.: 46 VH 13C5 CDR-H2 Residues52-67 of SEQ ID NO.: 46 VH 13C5 CDR-H3 Residues 100-112 of SEQ ID NO.:46 VL 13C5 CDR Set VL 13C5 CDR-L1 Residues 24-34 of SEQ ID NO.: 47 VL13C5 CDR-L2 Residues 50-56 of SEQ ID NO.: 47 VL 13C5 CDR-L3 Residues89-97 of SEQ ID NO.: 47 VH 29G5 CDR Set VH 29G5 CDR-H1 Residues 31-37 ofSEQ ID NO.: 48 VH 29G5 CDR-H2 Residues 52-67 of SEQ ID NO.: 48 VH 29G5CDR-H3 Residues 100-112 of SEQ ID NO.: 48 VL 29G5 CDR Set VL 29G5 CDR-L1Residues 24-34 of SEQ ID NO.: 49 VL 29G5 CDR-L2 Residues 50-56 of SEQ IDNO.: 49 VL 29G5 CDR-L3 Residues 89-97 of SEQ ID NO.: 49 VH 33C3 CDR SetVH 33C3 CDR-H1 Residues 31-37 of SEQ ID NO.: 50 VH 33C3 CDR-H2 Residues52-67 of SEQ ID NO.: 50 VH 33C3 CDR-H3 Residues 100-112 of SEQ ID NO.:50 VL 33C3 CDR Set VL 33C3 CDR-L1 Residues 24-34 of SEQ ID NO.: 51 VL33C3 CDR-L2 Residues 60-66 of SEQ ID NO.: 51 VL 33C3 CDR-L3 Residues89-97 of SEQ ID NO.: 51 VH 4A8 CDR Set VH 4A8 CDR-H1 Residues 31-35 ofSEQ ID NO.: 52 VH 4A8 CDR-H2 Residues 50-66 of SEQ ID NO.: 52 VH 4A8CDR-H3 Residues 99-107 of SEQ ID NO.: 52 VL 4A8 CDR Set VL 4A8 CDR-L1Residues 23-36 of SEQ ID NO.: 53 VL 4A8 CDR-L2 Residues 52-58 of SEQ IDNO.: 53 VL 4A8 CDR-L3 Residues 91-99 of SEQ ID NO.: 53 VH 1B6 CDR Set VH1B6 CDR-H1 Residues 31-35 of SEQ ID NO.: 54 VH 1B6 CDR-H2 Residues 50-65of SEQ ID NO.: 54 VH 1B6 CDR-H3 Residues 98-107 of SEQ ID NO.: 54 VL 1B6CDR Set VL 1B6 CDR-L1 Residues 24-38 of SEQ ID NO.: 55 VL 1B6 CDR-L2Residues 54-60 of SEQ ID NO.: 55 VL 1B6 CDR-L3 Residues 93-101 of SEQ IDNO.: 55 VH 3E5 CDR Set VH 3E5 CDR-H1 Residues 31-35 of SEQ ID NO.: 56 VH3E5 CDR-H2 Residues 50-65 of SEQ ID NO.: 56 VH 3E5 CDR-H3 Residues98-107 of SEQ ID NO.: 56 VL 3E5 CDR Set VL 3E5 CDR-L1 Residues 24-38 ofSEQ ID NO.: 57 VL 3E5 CDR-L2 Residues 54-60 of SEQ ID NO.: 57 VL 3E5CDR-L3 Residues 93-101 of SEQ ID NO.: 57 VH 6C8 CDR Set VH 6C8 CDR-H1Residues 31-35 of SEQ ID NO.: 58 VH 6C8 CDR-H2 Residues 50-65 of SEQ IDNO.: 58 VH 6C8 CDR-H3 Residues 98-107 of SEQ ID NO.: 58 VL 6C8 CDR SetVL 6C8 CDR-L1 Residues 24-38 of SEQ ID NO.: 59 VL 6C8 CDR-L2 Residues54-60 of SEQ ID NO.: 59 VL 6C8 CDR-L3 Residues 93-101 of SEQ ID NO.: 59VH 5D3 CDR Set VH 5D3 CDR-H1 Residues 31-35 of SEQ ID NO.: 60 VH 5D3CDR-H2 Residues 50-65 of SEQ ID NO.: 60 VH 5D3 CDR-H3 Residues 98-107 ofSEQ ID NO.: 60 VL 5D3 CDR Set VL 5D3 CDR-L1 Residues 24-38 of SEQ IDNO.: 61 VL 5D3 CDR-L2 Residues 54-60 of SEQ ID NO.: 61 VL 5D3 CDR-L3Residues 93-101 of SEQ ID NO.: 61 VH 8B6 CDR Set VH 8B6 CDR-H1 Residues31-35 of SEQ ID NO.: 62 VH 8B6 CDR-H2 Residues 50-65 of SEQ ID NO.: 62VH 8B6 CDR-H3 Residues 98-107 of SEQ ID NO.: 62 VL 8B6 CDR Set VL 8B6CDR-L1 Residues 24-38 of SEQ ID NO.: 63 VL 8B6 CDR-L2 Residues 54-60 ofSEQ ID NO.: 63 VL 8B6 CDR-L3 Residues 93-101 of SEQ ID NO.: 63 VH13C5.5L2E Set VH 13C5.5L2E CDR-H1 Residues 31-37 of SEQ ID NO.: 80 VH13C5.5L2E CDR-H2 Residues 52-67 of SEQ ID NO.: 80 VH 13C5.5L2E CDR-H3Residues 100-112 of SEQ ID NO.: 80 VL 13C5.5L2E Set VL 13C5.5L2E CDR-L1Residues 24-34 of SEQ ID NO.: 92 VL 13C5.5L2E CDR-L2 Residues 50-56 ofSEQ ID NO.: 92 VL 13C5.5L2E CDR-L3 Residues 89-97 of SEQ ID NO.: 92 VH13C5.5L2F Set VH 13C5.5L3F CDR-H1 Residues 31-37 of SEQ ID NO.: 80 VH13C5.5L3F CDR-H2 Residues 52-67 of SEQ ID NO.: 80 VH 13C5.5L3F CDR-H3Residues 100-112 of SEQ ID NO.: 80 VL 13C5.5L3F Set VL 13C5.5L3F CDR-L1Residues 24-34 of SEQ ID NO.: 93 VL 13C5.5L3F CDR-L2 Residues 50-56 ofSEQ ID NO.: 93 VL 13C5.5L2F CDR-L3 Residues 89-97 of SEQ ID NO.: 93 VH13C5.5L2EL3F Set VH 13C5.5L2EL3F CDR-H1 Residues 31-37 of SEQ ID NO.: 80VH 13C5.5L2EL3F CDR-H2 Residues 52-67 of SEQ ID NO.: 80 VH 13C5.5L2EL3FCDR-H3 Residues 100-112 of SEQ ID NO.: 80 VL 13C5.5L2EL3F Set VH13C5.5L2EL3F CDR-L1 Residues 24-34 of SEQ ID NO.: 94 VH 13C5.5L2EL3FCDR-L2 Residues 50-56 of SEQ ID NO.: 94 VH 13C5.5L2EL3F CDR-L3 Residues89-97 of SEQ ID NO.:
 94.


21. The host cell according to claim 20, which produces at least twovariable domain CDR sets selected from a group consisting of: VH 25C8CDR Set & VL 25C8 CDR Set; VH 9C11 CDR Set & VL 9C11 CDR Set; VH 21D9CDR Set & VL 21D9 CDR Set; VH 22D10 CDR Set & VL 22D10 CDR Set; VH 5F1CDR Set & VL 5F1 CDR Set; VH 5G1 CDR Set & VL 5G1 CDR Set; VH 3H7CDR Set& VL 3H7CDR Set; VH 14B2 CDR Set & VL 14B2 CDR Set; VH 13C5 CDR Set & VL13C5 CDR Set; VH 29G5 CDR Set & VL 29G5 CDR Set; VH 33C3 CDR Set & VL33C3 CDR Set; VH 4A8 CDR Set & VL 4A8 CDR Set; VH 1B6 CDR Set & VL 1B6CDR Set; VH 3E5CDR Set & VL 3E5 CDR Set; VH 6C8 CDR Set & VL 6C8 CDRSet; VH 5D3 CDR Set & VL 5D3 CDR Set; VH 8B6 CDR Set & VL 8B6 CDR Set;VH 13C5.5L2E CDR set & VL 13C5.5L2E CDR set; VH 13C5.5L3F CDR set & VL13C5.5L3F CDR set; and VH 13C5.5L2EL3F CDR set & VL 13C5.5L2EL3F CDRset.
 22. The isolated nucleic acid according to either claim 1 or claim2, further encoding a human acceptor framework.
 23. The isolated nucleicacid according to claim 22, wherein said human acceptor frameworkcomprises an amino acid sequence selected from the group consisting of:SEQ ID NO.: 6 SEQ ID NO.: 7 SEQ ID NO.: 8 SEQ ID NO.: 9 SEQ ID NO.: 10SEQ ID NO.: 11 SEQ ID NO.: 12 SEQ ID NO.: 13 SEQ ID NO.: 14 SEQ ID NO.:15 SEQ ID NO.: 16 SEQ ID NO.: 17 SEQ ID NO.: 18 SEQ ID NO.: 19 SEQ IDNO.: 20 SEQ ID NO.: 21 SEQ ID NO.: 22 SEQ ID NO.: 23 SEQ ID NO.: 24 SEQID NO.: 25 SEQ ID NO.: 26 SEQ ID NO.: 27 SEQ ID NO.: 28 SEQ ID NO.: 29SEQ ID NO.: 30 AND SEQ ID NO.:
 31.


24. The isolated nucleic acid according to claim 22, wherein said humanacceptor framework comprises at least one Framework Region amino acidsubstitution at a key residue, said key residue selected from the groupconsisting of: a residue adjacent to a CDR; a glycosylation siteresidue; a rare residue; a residue capable of interacting with humanIL-13; a residue capable of interacting with a CDR; a canonical residue;a contact residue between heavy chain variable region and light chainvariable region; a residue within a Vernier zone; and a residue in aregion that overlaps between a Chothia-defined variable heavy chain CDR1and a Kabat-defined first heavy chain framework.
 25. The isolatednucleic acid according to claim 24, wherein the key residue is selectedfrom the group consisting of 2L, 15L, 22L, 41L, 42L, 44L, 49L, 50L, 51L,62L, 71L, 73L, 10H, 44H, 46H, 48H, 67H, 68H, 70H, 72H, 74H, 76H, 83H,84H, 86H, 87H, and 97H.
 26. An isolated nucleic acid encoding an aminoacid sequence of at least one variable domain of an IL-13 bindingprotein, wherein said at least one variable domain comprises an aminoacid sequence selected from the group consisting of: SEQ ID NO.: 70 SEQID NO.: 71 SEQ ID NO.: 72 SEQ ID NO.: 73 SEQ ID NO.: 74 SEQ ID NO.: 75SEQ ID NO.: 76 SEQ ID NO.: 77 SEQ ID NO.: 78 SEQ ID NO.: 79 SEQ ID NO.:80 SEQ ID NO.: 81 SEQ ID NO.: 82 SEQ ID NO.: 83 SEQ ID NO.: 84 SEQ IDNO.: 85 SEQ ID NO.: 92 SEQ ID NO.: 93 and SEQ ID NO.:
 94.


27. The host cell according to claim 17, producing an IL-13 bindingprotein comprising two variable domains, wherein said two variabledomains comprise amino acid sequences selected from the group consistingof: SEQ ID NO.: 70 & SEQ ID NO.: 71, SEQ ID NO.: 72 & SEQ ID NO.: 73,SEQ ID NO.: 74 & SEQ ID NO.: 75, SEQ ID NO.: 76 & SEQ ID NO.: 77, SEQ IDNO.: 78 & SEQ ID NO.: 79, SEQ ID NO.: 80 & SEQ ID NO.: 81, SEQ ID NO.:82 & SEQ ID NO.: 83, SEQ ID NO.: 84 & SEQ ID NO.: 85 SEQ ID NO.: 80 &SEQ ID NO.: 92, SEQ ID NO.: 80 & SEQ ID NO.: 93, AND SEQ ID NO.: 80 &SEQ ID NO.:
 94.


28. The host cell according to claim 27, wherein said two variabledomains comprise amino acid sequences SEQ ID NO:80 and SEQ ID NO:81. 29.An isolated nucleic acid encoding a binding protein amino acid sequence,said binding protein being capable of binding IL-13 and comprising anantigen binding domain, said antigen binding domain comprising six CDRs:CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-H1comprises residues 31-37 of SEQ ID NO:80, CDR-H2 comprises residues52-67 of SEQ ID NO:80, CDR-H3 comprises residues 100-112 of SEQ IDNO:80, CDR-L1 comprises residues 24-34 of SEQ ID NO:81, CDR-L2 comprisesresidues 50-56 of SEQ ID NO:81, and CDR-L3 comprises residues 89-97 ofSEQ ID NO:81.
 30. The isolated nucleic according to claim 29, whereinsaid antigen binding domain comprises a heavy chain variable domaincomprising SEQ ID NO:80 and a light chain variable domain comprising SEQID NO:81.